Herbicide tolerant soybean plants and methods for identifying same

ABSTRACT

The invention provides specific transgenic soybean plants, plant material and seeds, characterized in that these products harbor a stack of specific transformation events at specific locations in the soybean genome (elite event EE-GM3 and elite event EE-GM2, or elite event EE-GM3 and elite event EE-GM1). The invention also provides for methods of producing soybean plants and seeds having elite event EE-GM3 and elite event EE-GM2, or elite event EE-GM3 and elite event EE-GM1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of U.S. Provisional PatentApplication No. 61/367,251, filed Jul. 23, 2010; U.S. Provisional PatentApplication No. 61/263,707, filed Nov. 23, 2009; and priority toEuropean Patent Application No. EP 09014565.7, filed Nov. 23, 2009, thedisclosures of each of which are hereby incorporated by reference intheir entireties.

FIELD OF THE INVENTION

This invention relates to transgenic soybean plants, plant material andseeds, characterized by harboring at least two specific transformationevents, particularly by the presence of at least two sets of genesencoding proteins that confer herbicide tolerance, each at a specificlocation in the soybean genome. The soybean plants of the inventioncombine the herbicide tolerance phenotype with an agronomic performance,genetic stability and functionality in different genetic backgroundsequivalent to the non-transformed soybean line in the absence ofherbicide(s). This invention further provides methods and kits foridentifying the presence of plant material comprising specificallytransformation event EE-GM3 and EE-GM2 or EE-GM1 in biological samples.

BACKGROUND OF THE INVENTION

The phenotypic expression of a transgene in a plant is determined bothby the structure of the gene or genes itself and by its or theirlocation in the plant genome. At the same time the presence of thetransgenes or “foreign DNA” at different locations in the genome willinfluence the overall phenotype of the plant in different ways. Theagronomically or industrially successful introduction of a commerciallyinteresting trait in a plant by genetic manipulation can be a lengthyprocedure dependent on different factors. The actual transformation andregeneration of genetically transformed plants are only the first in aseries of selection steps, which include extensive geneticcharacterization, breeding, and evaluation in field trials, eventuallyleading to the selection of an elite event.

The unequivocal identification of an elite event is becomingincreasingly important in view of discussions on Novel Food/Feed,segregation of GMO and non-GMO products and the identification ofproprietary material. Ideally, such identification method is both quickand simple, without the need for an extensive laboratory set-up.Furthermore, the method should provide results that allow unequivocaldetermination of the elite event without expert interpretation, butwhich hold up under expert scrutiny if necessary. Specific tools for usein the identification of elite event EE-GM3 and EE-GM1 or EE-GM2 inbiological samples are described herein.

In this invention, EE-GM3 has been identified as an elite event from apopulation of transgenic soybean plants in the development of herbicidetolerant soybean (Glycine max) comprising a gene coding for glyphosatetolerance combined with a gene conferring tolerance to 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors, each under control of aplant-expressible promoter.

EE-GM1 and EE-GM2 have previously been identified as elite events from apopulation of transgenic soybean plants in the development of herbicidetolerant soybean (Glycine max) comprising a gene coding for glufosinatetolerance under control of a plant-expressible promoter and aredescribed in WO2006/108674 and WO2006/108675 (both publications hereinincorporated by reference).

Soybean plants comprising a herbicide tolerance gene have been disclosedin the art. However, none of the prior art disclosures teach or suggestthe present invention.

It is known in the art that getting a commercial herbicide tolerantelite transformation event in soybean plants with acceptable agronomicperformance, with no yield drag, and providing sufficient herbicidetolerance, certainly to 3 different classes of herbicides, is by nomeans straightforward.

Indeed, it has been reported that the first soybean event (event 40-3-2)released on the market with herbicide tolerance, had a significant yielddrag compared to (near-)isogenic lines (Elmore et al. (2001) Agron. J.93:408-412).

Also, Optimum™ GAT™ soybeans (event 356043) were developed to combinetolerance to glyphosate with tolerance to ALS herbicides, but it hasbeen reported that these soybeans were not meeting the standards forglyphosate tolerance by itself (without combination with anotherglyphosate tolerance soybean event (such as event 40-3-2).

SUMMARY OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention relates to a transgenic soybean plant, or seed,cells or tissues thereof, comprising, stably integrated into its genome,an expression cassette which comprises a herbicide tolerance genecomprising the coding sequence of the 2mEPSPS gene and another herbicidetolerance gene comprising the coding sequence of HPPD-PF W336 (both asdescribed in Example 1.1 herein and as represented in SEQ ID No 1) aswell as a second expression cassette comprising a herbicide tolerancegene comprising the coding sequence of a phosphinothricin acetyltransferase, as described in Example 1 herein and as represented in SEQID No 11. The transgenic soybean plant, or seed, cells or tissuesthereof are tolerant to herbicides based on glyphosate, based onglufosinate, and an HPPD inhibitor herbicide such as isoxaflutole, and,in the absence of herbicide(s), has an agronomic performance which issubstantially equivalent to the non-transgenic isogenic line. Afterapplication of one or more herbicides to which tolerance is provided,the plant will have a superior agronomic phenotype compared to anon-transgenic plant.

According to the present invention the soybean plant or seed, cells ortissues thereof comprise elite event EE-GM3 and EE-GM1 or EE-GM3 andEE-GM2.

More specifically, the present invention relates to a transgenic soybeanplant, seed, cells or tissues thereof, the genomic DNA of which ischaracterized by the fact that, when analyzed in a PCR IdentificationProtocol as described herein, using two primers directed to the 5′ or 3′flanking region of EE-GM3 and the foreign DNA comprising herbicidetolerance genes in EE-GM-3, respectively, yields a fragment which isspecific for EE-GM3 and further when analyzed in a PCR IdentificationProtocol as described herein, using two primers directed to the 5′ or 3′flanking region of EE-GM1 or EE-GM2 and the foreign DNA comprisingglufosinate tolerance genes, respectively, yields a fragment which isspecific for EE-GM1 or EE-GM2. The primers for detection of EE-GM3 maybe directed against the 5′ flanking region within SEQ ID NO: 2 and theforeign DNA comprising herbicide tolerance genes, respectively. Theprimers for detection of EE-GM3 may also be directed against the 3′flanking region within SEQ ID NO: 3 and the foreign DNA comprisingherbicide tolerance genes, respectively, such as the primers comprisingor consisting (essentially) of the nucleotide sequence of SEQ ID NO: 5and SEQ ID NO: 4 or SEQ ID No.: 7 respectively, and yield a DNA fragmentof between 100 and 800 bp, such as a fragment of about 263 bp or about706 bp. The primers for detection of EE-GM1 may be directed against the5′ flanking region within SEQ ID NO: 12 and the foreign DNA comprisingherbicide tolerance genes, respectively. The primers for detection ofEE-GM1 may also be directed against the 3′ flanking region within SEQ IDNO: 13 and the foreign DNA comprising herbicide tolerance genes,respectively, such as the primers comprising or consisting (essentially)of the nucleotide sequence of SEQ ID NO: 16 and SEQ ID NO: 17respectively, and yield a DNA fragment of between 100 and 500 bp, suchas a fragment of about 183 bp. The primers for detection of EE-GM2 maybe directed against the 5′ flanking region within SEQ ID NO: 14 and theforeign DNA comprising herbicide tolerance genes, respectively. Theprimers for detection of EE-GM2 may also be directed against the 3′flanking region within SEQ ID NO: 15 and the foreign DNA comprisingherbicide tolerance genes, respectively, such as the primers comprisingor consisting (essentially) of the nucleotide sequence of SEQ ID NO: 18and SEQ ID NO: 19 respectively, and yield a DNA fragment of between 100and 500 bp, such as a fragment of about 151 bp.

Reference seed comprising the elite event EE-GM3 of the invention hasbeen deposited at the NCIMB under accession number NCIMB 41659.Reference seed comprising the elite event EE-GM1 of the invention hasbeen deposited at the NCIMB under accession number NCIMB 41658.Reference seed comprising the elite event EE-GM2 of the invention hasbeen deposited at the NCIMB under accession number NCIMB 41660.Reference seed comprising elite event EE-GM3 and EE-GM1 was deposited atthe ATCC under accession number PTA-11041. Reference seed comprisingelite event EE-GM3 and EE-GM2 was deposited at the ATCC under accessionnumber PTA-11042.

One embodiment of the invention is seed comprising elite event EE-GM3(reference seed comprising said event being deposited as NCIMB accessionnumber NCIMB 41659) and further comprising elite event EE-GM1 (referenceseed comprising said event being deposited as NCIMB accession numberNCIMB 41658), or seed comprising event EE-GM3 and EE-GM1 (reference seedcomprising said events being deposited at the ATCC under accessionnumber PTA-11041) which will grow into a soybean plant tolerant to atleast three herbicides, particularly tolerant to glyphosate and/or HPPDinhibitors such as isoxaflutole and/or glutamine synthetase inhibitorssuch as glufosinate.

Another embodiment of the invention is seed comprising elite eventEE-GM3 (reference seed comprising said event being deposited as NCIMBaccession number NCIMB 41659) and further comprising elite event EE-GM2(reference seed comprising said event being deposited as NCIMB accessionnumber NCIMB 41660), or seed comprising event EE-GM3 and EE-GM2(reference seed comprising said events being deposited at the ATCC underaccession number PTA-11042), which will grow into a soybean planttolerant to at least three herbicides, particularly tolerant toglyphosate and/or HPPD inhibitors such as isoxaflutole and/or glutaminesynthetase inhibitors such as glufosinate.

The seed of NCIMB deposit number NCIMB 41659, is a seed lot consistingof at least about 95% transgenic seeds, comprising elite event EE-GM3,which will grow into herbicide tolerant plants, whereby the plants areglyphosate and/or isoxaflutole tolerant plants. The seed or progeny seedobtainable or obtained from the deposited seed (e.g., following crossingwith other soybean plants with a different genetic background) can besown and the growing plants can be treated with glyphosate or HPPDinhibitors such as isoxaflutole as described herein to obtain glyphosateor isoxaflutole-tolerant plants, comprising elite event EE-GM3. The seedof NCIMB deposit number NCIMB 41658 and NCIMB 41660 are seed lotsconsisting of at least about 95% transgenic seeds, comprising eliteevents EE-GM1 or EE-GM2, respectively, which will grow into herbicidetolerant plants, whereby the plants are glufosinate-tolerant plants. Theseed can be sown and the growing plants can be treated with glufosinateas described herein to obtain glufosinate tolerant plants, comprisingthe elite events EE-GM1 or EE-GM2.

The seed of ATCC deposit number PTA-11041, is a seed lot consisting ofat least about 95% transgenic seeds, comprising elite event EE-GM3 andelite event EE-GM1 in homozygous form, which will grow into herbicidetolerant plants, whereby the plants are glyphosate, glufosinate and/orisoxaflutole tolerant plants. The seed or progeny seed obtainable orobtained from the deposited seed (e.g., following crossing with othersoybean plants with a different genetic background) can be sown and thegrowing plants can be treated with glyphosate, glufosinate and/or HPPDinhibitors such as isoxaflutole, as described herein to obtainglyphosate, glufosinate and/or isoxaflutole-tolerant plants, comprisingelite event EE-GM3 and EE-GM1.

The seed of ATCC deposit number PTA-11042, is a seed lot consisting ofat least about 95% transgenic seeds, comprising elite event EE-GM3 andelite event EE-GM2 in homozygous form, which will grow into herbicidetolerant plants, whereby the plants are glyphosate, glufosinate and/orisoxaflutole tolerant plants. The seed or progeny seed obtainable orobtained from the deposited seed (e.g., following crossing with othersoybean plants with a different genetic background) can be sown and thegrowing plants can be treated with glyphosate, glufosinate and/or HPPDinhibitors such as isoxaflutole, as described herein to obtainglyphosate, glufosinate and/or isoxaflutole-tolerant plants, comprisingelite event EE-GM3 and EE-GM2.

The invention further relates to cells, tissues, progeny, anddescendants from a plant comprising the elite event EE-GM3 and furthercomprising elite event EE-GM1, and which may be obtained by growing aplant comprising the elite event EE-GM3 and EE-GM1 deposited at ATCC asPTA-11041, or by growing a plant comprising the elite event EE-GM3 fromthe seed deposited at the NCIMB having accession number NCIMB 41659 andcrossing said plant with a plant comprising elite event EE-GM1 grownfrom the seed deposited at the NCIMB having accession number NCIMB41658. The invention also relates to cells, tissues, progeny, anddescendants from a plant comprising the elite event EE-GM3 and furthercomprising elite event EE-GM2, and which may be obtained by growing aplant comprising the elite event EE-GM3 and EE-GM2 deposited at ATCC asPTA-11042, or by growing a plant comprising the elite event EE-GM3 fromthe seed deposited at the NCIMB having accession number NCIMB 41659 andcrossing said plant with a plant comprising elite event EE-GM2 grownfrom the seed deposited at the NCIMB having accession number NCIMB41660. The invention further relates to plants obtainable from (such asby propagation of and/or breeding with) a soybean plant or seed asdescribed immediately above. The invention also relates to soybeanplants comprising elite event EE-GM3 and EE-GM1 or EE-GM3 and EE-GM2.

The invention further relates to a method for identifying a transgenicplant, or cells or tissues thereof, comprising elite event EE-GM3 andEE-GM1 or EE-GM3 and EE-GM2, which method is based on identifying thepresence of characterizing DNA sequences or amino acids encoded by suchDNA sequences in the transgenic plant, cells or tissues. According to apreferred embodiment of the invention, such characterizing DNA sequencesare sequences of at least 15 bp, 15 bp, at least 20 bp, 20 bp, or 30 bpor more which comprise the insertion site of the event, i.e., both apart of the inserted foreign DNA comprising herbicide tolerance genesand a part of the soybean genome (either the 5′ or 3′ flanking region)contiguous therewith, allowing specific identification of the eliteevent.

The present invention further relates to methods for identifying eliteevents EE-GM3 and EE-GM1 or elite events EE-GM3 and EE-GM2 in biologicalsamples, which methods are based on primers or probes which specificallyrecognize the 5′ and/or 3′ flanking sequence of the foreign DNAcomprising the herbicide tolerance genes in EE-GM3 and EE-GM1 or EE-GM3and EE-GM2.

More specifically, the invention relates to a method comprisingamplifying two sequences of a nucleic acid present in biologicalsamples, using two polymerase chain reactions each with at least twoprimers or one polymerase chain reaction with at least four primers, thefirst primer recognizing the 5′ or 3′ flanking region of foreign DNAcomprising herbicide tolerance genes comprising the herbicide tolerancegenes in EE-GM3, the second primer recognizing a sequence within theforeign DNA comprising herbicide tolerance genes of EE-GM3, the thirdprimer recognizing the 5′ or 3′ flanking region of foreign DNAcomprising the herbicide tolerance genes in EE-GM1 or EE-GM2, and thefourth primer recognizing a sequence within the foreign DNA comprisingthe herbicide tolerance genes of EE-GM1 or EE-GM2, preferably to obtaintwo DNA fragments of between 100 and 800 bp. The primers for identifyingEE-GM3 may recognize a sequence within the 5′ flanking region of EE-GM3(SEQ ID No. 2, from position 1 to position 1451) or within the 3′flanking region of EE-GM3 (complement of SEQ ID No 3 from position 241to position 1408) and a sequence within the foreign DNA comprising theherbicide tolerance genes (complement of SEQ ID No 2 from position 1452to 1843 or SEQ ID No 3 from position 1 to position 240, or SEQ ID No 20from nucleotide position 1452 to nucleotide position 16638 or itscomplement), respectively. The primer recognizing the 3′ flanking regionmay comprise the nucleotide sequence of SEQ ID No. 5 and the primerrecognizing a sequence within the foreign DNA comprising herbicidetolerance genes may comprise the nucleotide sequence of SEQ ID No. 4 orSEQ ID No.: 7 described herein. The primers for identifying EE-GM1 mayrecognize a sequence within the 5′ flanking region of EE-GM1 (SEQ ID No.12, from position 1 to position 209) or within the 3′ flanking region ofEE-GM1 (complement of SEQ ID No 13 from position 569 to position 1000)and a sequence within the foreign DNA comprising herbicide tolerancegene of EE-GM1 (complement of SEQ ID No 12 from position 210 to 720, orSEQ ID No 13 from position 1 to position 568), respectively. The primerrecognizing the 5′ flanking region of EE-GM1 may comprise the nucleotidesequence of SEQ ID No. 16 and the primer recognizing a sequence withinthe foreign DNA of EE-GM1 may comprise the nucleotide sequence of SEQ IDNo. 17 described herein. The primers for identifying EE-GM2 mayrecognize a sequence within the 5′ flanking region of EE-GM2 (SEQ ID No.14, from position 1 to position 311) or within the 3′ flanking region ofEE-GM2 (complement of SEQ ID No 15 from position 508 to position 1880)and a sequence within the foreign DNA comprising herbicide tolerancegene of EE-GM1 (complement of SEQ ID No 14 from position 312 to 810, orSEQ ID No 15 from position 1 to position 507), respectively. The primerrecognizing the 3′ flanking region of EE-GM2 may comprise the nucleotidesequence of SEQ ID No. 18 and the primer recognizing a sequence withinthe foreign DNA of EE-GM2 may comprise the nucleotide sequence of SEQ IDNo. 19 described herein. The PCR amplification may be donesimultaneously or sequentially.

The present invention more specifically relates to a method foridentifying elite event EE-GM3 and EE-GM1 in biological samples, whichmethod comprises amplifying at least two sequences of nucleic acidspresent in a biological sample, using a polymerase chain reaction withtwo primers comprising or consisting (essentially) of the nucleotidesequence of SEQ ID No. 4 and SEQ ID No. 5, respectively, to obtain a DNAfragment of about 263 bp, or with two primers comprising or consisting(essentially) of the nucleotide sequence of SEQ ID No. 5 and SEQ ID No.7 respectively, to obtain a DNA fragment of about 706 bp, and apolymerase chain reaction with two primers comprising or consisting(essentially) of the nucleotide sequence of SEQ ID No. 16 and SEQ ID No.17 respectively, to obtain a DNA fragment of about 183 bp. The twopolymerase chain reactions may be performed simultaneously orsequentially.

The present invention more specifically relates to a method foridentifying elite event EE-GM3 and EE-GM2 in biological samples, whichmethod comprises amplifying at least two sequences of nucleic acidspresent in a biological sample, using a polymerase chain reaction withtwo primers comprising or consisting (essentially) of the nucleotidesequence of SEQ ID No. 4 and SEQ ID No. 5 respectively, to obtain a DNAfragment of about 263 bp, or with two primers comprising or consisting(essentially) of the nucleotide sequence of SEQ ID No. 5 and SEQ ID No.7 respectively, to obtain a DNA fragment of about 706 bp, and apolymerase chain reaction with two primers comprising or consisting(essentially) of the nucleotide sequence of SEQ ID No. 18 and SEQ ID No.19 respectively, to obtain a DNA fragment of about 151 bp. The twopolymerase chain reactions may be performed simultaneously orsequentially.

The present invention further relates to the specific flanking sequencesof EE-GM3 described herein in combination with the specific flankingsequences of EE-GM1, which can be used to develop specificidentification methods for simultaneous presence of EE-GM3 and EE-GM1 inbiological samples. Such combined specific flanking sequences may alsobe used as reference control material in identification assays. Moreparticularly, the invention relates to the 5′ and/or 3′ flanking regionsof EE-GM3 in combination with the 5′ and/or 3′ flanking regions ofEE-GM1 which can be used for the development of specific primers andprobes as further described herein. Also suitable as reference materialare nucleic acid molecules, preferably of about 150-850 bp, comprisingthe sequence which can be amplified by primers comprising or consisting(essentially) of the nucleotide sequence of SEQ ID No. 7 and SEQ ID No.5 or of SEQ ID No. 4 and SEQ ID No. 5 in combination with nucleic acidmolecules comprising the sequence which can be amplified by primerscomprising or consisting (essentially) of the nucleotide sequence of SEQID No. 16 and SEQ ID No. 17, particularly such nucleic acid moleculesare obtained using such primers in material comprising EE-GM3 andEE-GM1.

The present invention further also relates to the specific flankingsequences of EE-GM3 described herein in combination with the specificflanking sequence of EE-GM2, which can be used to develop specificidentification methods for simultaneous presence of EE-GM3 and EE-GM2 inbiological samples. Such combined specific flanking sequences may alsobe used as reference control material in identification assays. Moreparticularly, the invention relates to the 5′ and/or 3′ flanking regionsof EE-GM3 in combination with the 5′ and/or 3′ flanking regions ofEE-GM2 which can be used for the development of specific primers andprobes as further described herein. Also suitable as reference materialare nucleic acid molecules, preferably of about 150-850 bp, comprisingthe sequence which can be amplified by primers comprising or consisting(essentially) of the nucleotide sequence of SEQ ID No. 7 and SEQ ID No.5 or of SEQ ID No. 4 and SEQ ID No. 5 in combination with nucleic acidmolecules comprising the sequence which can be amplified by primerscomprising or consisting (essentially) of the nucleotide sequence of SEQID No. 18 and SEQ ID No. 19, particularly such nucleic acid moleculesare obtained using such primers in material comprising EE-GM3 andEE-GM2.

The invention further relates to identification methods for thesimultaneous presence of EE-GM3 and EE-GM1 in biological samples basedon the use of such specific primers or probes. Primers for EE-GM3detection may comprise, consist or consist essentially of a nucleotidesequence of 17 to about 200 consecutive nucleotides selected from thenucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 1451or the complement of the nucleotide sequence of SEQ ID 3 from nucleotide241 to nucleotide 1408, combined with primers comprising, consisting, orconsisting essentially of a nucleotide sequence of 17 to about 200consecutive nucleotides selected from the nucleotide sequence of SEQ IDNo 1, such as a nucleotide sequence of 17 to about 200 consecutivenucleotides selected from the complement of the nucleotide sequence ofSEQ ID No 2 from nucleotide 1452 to nucleotide 1843 or the nucleotidesequence of SEQ ID No 3 from nucleotide 1 to nucleotide 240. Primers forEE-GM1 detection may comprise, consist or consist essentially of anucleotide sequence of 17 to about 200 consecutive nucleotides selectedfrom the nucleotide sequence of SEQ ID No 12 from nucleotide 1 tonucleotide 209 or the complement of the nucleotide sequence of SEQ ID 13from nucleotide 569 to nucleotide 1000, combined with primerscomprising, consisting, or consisting essentially of a nucleotidesequence of 17 to about 200 consecutive nucleotides selected from thenucleotide sequence of SEQ ID No 11, such as a nucleotide sequence of 17to about 200 consecutive nucleotides selected from the complement of thenucleotide sequence of SEQ ID No 12 from nucleotide 219 to nucleotide720 or the nucleotide sequence of SEQ ID No 13 from nucleotide 1 tonucleotide 568. Primers may also comprise these nucleotide sequenceslocated at their extreme 3′ end, and further comprise unrelatedsequences or sequences derived from the mentioned nucleotide sequences,but comprising mismatches.

The invention further relates to identification methods for thesimultaneous presence of EE-GM3 and EE-GM2 in biological samples basedon the use of such specific primers or probes. Primers for EE-GM3detection may comprise, consist or consist essentially of a nucleotidesequence of 17 to about 200 consecutive nucleotides as described in theprevious paragraph. Primers for EE-GM2 detection may comprise, consistor consist essentially of a nucleotide sequence of 17 to about 200consecutive nucleotides selected from the nucleotide sequence of SEQ IDNo 14 from nucleotide 1 to nucleotide 311 or the complement of thenucleotide sequence of SEQ ID 15 from nucleotide 508 to nucleotide 1880,combined with primers comprising, consisting or consisting essentiallyof a nucleotide sequence of 17 to about 200 consecutive nucleotidesselected from the nucleotide sequence of SEQ ID No 11, such as anucleotide sequence of 17 to about 200 consecutive nucleotides selectedfrom the complement of the nucleotide sequence of SEQ ID No 14 fromnucleotide 312 to nucleotide 810 or the nucleotide sequence of SEQ ID No15 from nucleotide 1 to nucleotide 507. Primers may also comprise thesenucleotide sequences located at their extreme 3′ end, and furthercomprise unrelated sequences or sequences derived from the mentionednucleotide sequences, but comprising mismatches.

The invention further relates to kits for identifying elite eventsEE-GM3 and EE-GM1 in biological samples, said kits comprising at leastone primer or probe which specifically recognizes the 5′ or 3′ flankingregion of the foreign DNA comprising herbicide tolerance genes in EE-GM3and at least one primer or probe which specifically recognizes the 5′ or3′ flanking region of the foreign DNA comprising herbicide tolerancegenes in EE-GM1.

The invention further relates to kits for identifying elite eventsEE-GM3 and EE-GM2 in biological samples, said kits comprising at leastone primer or probe which specifically recognizes the 5′ or 3′ flankingregion of the foreign DNA comprising herbicide tolerance genes in EE-GM3and at least one primer or probe which specifically recognizes the 5′ or3′ flanking region of the foreign DNA comprising a herbicide tolerancegene in EE-GM2.

The kits of the invention may comprise, in addition to a primer whichspecifically recognizes the 5′ or 3′ flanking region of EE-GM3 and the5′ or 3′ flanking region of EE-GM1, a further primer which specificallyrecognizes a sequence within the foreign DNA comprising herbicidetolerance genes of EE-GM3 and a further primer which specificallyrecognizes a sequence within the foreign DNA comprising herbicidetolerance genes of EE-GM1, for use in a PCR Identification Protocol.

The kits of the invention may also comprise, in addition to a primerwhich specifically recognizes the 5′ or 3′ flanking region of EE-GM3 andthe 5′ or 3′ flanking region of EE-GM2, a further primer whichspecifically recognizes a sequence within the foreign DNA comprisingherbicide tolerance genes of EE-GM3 and a further primer whichspecifically recognizes a sequence within the foreign DNA comprisingherbicide tolerance genes of EE-GM2, for use in a PCR IdentificationProtocol.

The primer recognizing the 3′ flanking region of EE-GM3 may comprise thenucleotide sequence of SEQ ID No. 5 and the primer recognizing thetransgenes or foreign DNA comprising herbicide tolerance genes of EE-GM3may comprises the nucleotide sequence of SEQ ID No. 4 or 7, or any otherprimer or primer combination for EE-GM3 detection as described herein,in combination with a primer recognizing the 5′ flanking region ofEE-GM1 which may comprise the nucleotide sequence of SEQ ID No. 16 and aprimer recognizing the transgenes of foreign DNA comprising theherbicide tolerance gene of EE-GM1 may comprise the nucleotide sequenceof SEQ ID No 17.

The primer recognizing the 3′ flanking region of EE-GM3 may comprise thenucleotide sequence of SEQ ID No. 5 and the primer recognizing thetransgenes or foreign DNA comprising herbicide tolerance genes of EE-GM3may comprises the nucleotide sequence of SEQ ID No. 4 or 7, or any otherprimer or primer combination for EE-GM3 detection as described herein,in combination with a primer recognizing the 5′ flanking region ofEE-GM2 which may comprise the nucleotide sequence of SEQ ID No. 18 and aprimer recognizing the transgenes of foreign DNA comprising theherbicide tolerance gene of EE-GM2 may comprise the nucleotide sequenceof SEQ ID No 19.

The invention further relates to a kit for identifying elite eventEE-GM3 and EE-GM1 in biological samples, said kit comprising PCR primerscomprising or consisting (essentially) of the nucleotide sequence of SEQID No. 5 and SEQ ID No. 4 and PCR primers comprising or consisting(essentially) of the nucleotide sequence of SEQ ID 16 and SEQ ID 17 foruse in the EE-GM3/EE-GM1 PCR based identification.

The invention further relates to a kit for identifying elite eventEE-GM3 and EE-GM2 in biological samples, said kit comprising PCR primerscomprising or consisting (essentially) of the nucleotide sequence of SEQID No. 5 and SEQ ID No. 4 and PCR primers comprising or consisting(essentially) of the nucleotide sequence of SEQ ID 18 and SEQ ID 19 foruse in the EE-GM3/EE-GM2 PCR based identification.

The invention also relates to a kit for identifying elite event EE-GM3and EE-GM1 in biological samples, which kit comprises two specificprobes, a first probe comprising or consisting (essentially) of asequence which corresponds (or is complementary to) a sequence havingbetween 80% and 100% sequence identity with a specific region of EE-GM3and a second probe comprising or consisting (essentially) of a sequencewhich corresponds (or is complementary to) a sequence having between 80%and 100% sequence identity with a specific region of EE-GM1. Preferably,the sequence of the first probe corresponds to a specific regioncomprising part of the 5′ or 3′ flanking region of EE-GM3 and the secondprobe corresponds to a specific region comprising part of the 5′ or 3′flanking region of EE-GM1. Most preferably the EE-GM3 specific probecomprises or consists (essentially) of (or is complementary to) asequence having between 80% and 100% sequence identity to the sequencebetween nucleotide 1441 to 1462 of SEQ ID No 2 or a sequence havingbetween 80% and 100% sequence identity to the sequence betweennucleotide 220 to 260 of ID No. 3 and the EE-GM1 specific probecomprises, consists (essentially) of (or is complementary to) a sequencehaving between 80% and 100% sequence identity to the sequence betweennucleotide 199 to 220 of SEQ ID No 12 or a sequence having between 80%and 100% sequence identity to the sequence between nucleotide 558 to 579of ID No. 13.

The invention also relates to a kit for identifying elite event EE-GM3and EE-GM2 in biological samples, which kit comprises two specificprobes, a first probe comprising or consisting (essentially) of asequence which corresponds (or is complementary to) a sequence havingbetween 80% and 100% sequence identity with a specific region of EE-GM3and a second probe comprising or consisting (essentially) of a sequencewhich corresponds (or is complementary to) a sequence having between 80%and 100% sequence identity with a specific region of EE-GM2. Preferably,the sequence of the first probe corresponds to a specific regioncomprising part of the 5′ or 3′ flanking region of EE-GM3 and the secondprobe corresponds to a specific region comprising part of the 5′ or 3′flanking region of EE-GM2. Most preferably the EE-GM3 specific probecomprises or consists (essentially) of (or is complementary to) asequence having between 80% and 100% sequence identity to the sequencebetween nucleotide 1441 to 1462 of SEQ ID No 2 or a sequence havingbetween 80% and 100% sequence identity to the sequence betweennucleotide 220 to 260 of ID No. 3 and the EE-GM2 specific probecomprises or consists (essentially) of (or is complementary to) asequence having between 80% and 100% sequence identity to the sequencebetween nucleotide 301 to 322 of SEQ ID No 14 or a sequence havingbetween 80% and 100% sequence identity to the sequence betweennucleotide 497 to 518 of ID No. 15.

According to another aspect of the invention, DNA sequences aredisclosed comprising a junction site of the event and sufficient lengthof polynucleotides of both the soybean genomic DNA and the foreign DNAcomprising herbicide tolerance genes (transgene) of each event, so as tobe useful as primer or probe for the detection of EE-GM3 and EE-GM1.Such sequences may comprise at least 9 nucleotides of the soybeangenomic DNA and a similar number of nucleotides of the foreign DNAcomprising herbicide tolerance genes (transgene) of EE-GM3 or EE-GM1, ateach side of the junction site respectively. Most preferably, such DNAsequences comprise at least 9 nucleotides of the soybean genomic DNA anda similar number of nucleotides of the foreign DNA comprising herbicidetolerance genes contiguous with the junction site in SEQ ID NO: 2 or SEQID NO: 3 and at least 9 nucleotides of the soybean genomic DNA and asimilar number of nucleotides of the foreign DNA comprising herbicidetolerance genes contiguous with the junction site in SEQ ID NO: 12 orSEQ ID NO: 13.

According to yet another aspect of the invention, DNA sequences aredisclosed comprising a junction site of the event and sufficient lengthof polynucleotides of both the soybean genomic DNA and the foreign DNAcomprising herbicide tolerance genes (transgene) of each event, so as tobe useful as primer or probe for the detection of EE-GM3 and EE-GM2.Such sequences may comprise at least 9 nucleotides of the soybeangenomic DNA and a similar number of nucleotides of the foreign DNAcomprising herbicide tolerance genes (transgene) of EE-GM3 or EE-GM2, ateach side of the insertion site respectively. Most preferably, such DNAsequences comprise at least 9 nucleotides of the soybean genomic DNA anda similar number of nucleotides of the foreign DNA comprising herbicidetolerance genes contiguous with the junction site in SEQ ID NO: 2 or SEQID NO: 3 and at least 9 nucleotides of the soybean genomic DNA and asimilar number of nucleotides of the foreign DNA comprising herbicidetolerance genes contiguous with the junction site in SEQ ID NO: 14 orSEQ ID NO: 15.

The methods and kits encompassed by the present invention can be usedfor different purposes such as, but not limited to the following: toidentify the presence or determine the (lower) threshold of EE-GM3 andEE-GM1 or of EE-GM3 and EE-GM2 in plants, plant material or in productssuch as, but not limited to food or feed products (fresh or processed)comprising or derived from plant material; additionally oralternatively, the methods and kits of the present invention can be usedto identify transgenic plant material for purposes of segregationbetween transgenic and non-transgenic material; additionally oralternatively, the methods and kits of the present invention can be usedto determine the quality (i.e., percentage pure material) of plantmaterial comprising EE-GM3 and EE-GM1 or EE-GM3 and EE-GM1.

The invention further relates to the 5′ and/or 3′ flanking regions ofEE-GM3 GM3 in combination with specific primers and probes developedfrom the 5′ and/or 3′ flanking sequences of EE-GM1 or EE-GM2, as well asto the specific primers and probes developed from the 5′ and/or 3′flanking sequences of EE-GM3 in combination with specific primers andprobes developed from the 5′ and/or 3′ flanking sequences of EE-GM1 orEE-GM2.

The invention also relates to genomic DNA obtained from plantscomprising elite events EE-GM3 and EE-GM1 or plants comprising eliteevents EE-GM3 and EE-GM2. Such genomic DNA may be used as referencecontrol material in the identification assays herein described.

Also provided herein is a transgenic herbicide tolerant soybean plant,or cells, parts, seeds or progeny thereof, each comprising at least twoelite events, said first elite event comprising a foreign DNAcomprising:

-   -   i) a first chimeric gene which comprises a modified epsps gene        from Zea mays encoding a glyphosate tolerant EPSPS enzyme under        the control of a plant-expressible promoter, and    -   ii) a second chimeric gene which comprises a modified hppd gene        from Pseudomonas fluorescens encoding an HPPD inhibitor        herbicide tolerant enzyme under the control of a        plant-expressible promoter, and said second elite event        comprises a (third) chimeric gene which comprises a modified        glufosinate tolerance gene derived from Streptomyces        viridochromogenes encoding a glufosinate (or phosphinothricin)        acetyltransferase enzyme under the control of a        plant-expressible promoter.

In one embodiment, said first elite event comprises nucleotides 1 to1451 of SEQ ID No 2 immediately upstream of and contiguous with saidforeign DNA and nucleotides 241 to 1408 of SEQ ID No 3 immediatelydownstream of and contiguous with said foreign DNA, and said secondevent comprises nucleotides 1 to 209 of SEQ ID No 12 immediatelyupstream of and contiguous with said foreign DNA and nucleotides 569 to1000 of SEQ ID No 13 immediately downstream of and contiguous with saidforeign DNA, or said second event comprises nucleotides 1 to 311 of SEQID No 14 immediately upstream of and contiguous with said foreign DNAand nucleotides 508 to 1880 of SEQ ID No 15 immediately downstream ofand contiguous with said foreign DNA.

In a further embodiment, said elite event is obtainable by breeding witha soybean plant grown from reference seed comprising said events havingbeen deposited at the ATCC under deposit number PTA-11041 or PTA-11042,or obtainable from reference seed comprising said first event havingbeen deposited at NCIMB under accession number NCIMB 41659, andobtainable from reference seed comprising said second event having beendeposited at NCIMB under accession number NCIMB 41658 or NCIMB accessionnumber NCIMB 41660.

In another embodiment, the genomic DNA of said soybean plant, or cells,parts, seeds or progeny thereof when analyzed using the elite eventidentification protocol for said first elite event with two primerscomprising the nucleotide sequence of SEQ ID No 4 and SEQ ID No 5respectively, yields a DNA fragment of about 263 by or 263 bp, and whenanalyzed using the elite event identification protocol for said secondelite event with two primers comprising the nucleotide sequence of SEQID No 16 and SEQ ID No 17 respectively, yields a DNA fragment of about183 bp or 183 bp, or with two primers comprising the nucleotide sequenceof SEQ ID No 18 and SEQ ID No 19 respectively, yields a DNA fragment ofabout 151 bp or 151 bp.

Also provided herein is a method for identifying a transgenic soybeanplant, or cells, parts, seed or progeny thereof comprising 2 eliteevents, wherein said plant, cells, seed, or progeny are tolerant toglyphosate, glufosinate and an HPPD inhibitor herbicide (such asisoxaflutole), in biological samples, said method comprising amplifyinga DNA fragment of between 100 and 500 bp from a nucleic acid of saidfirst event present in biological samples using a polymerase chainreaction with at least two primers, one of said primers recognizing the5′ flanking region of the first elite event specified above, said 5′flanking region comprising the nucleotide sequence of SEQ ID No 2 fromnucleotide 1 to nucleotide 1451, or the 3′ flanking region of said firstelite event, said 3′ flanking region comprising the nucleotide sequenceof the complement of SEQ ID No 3 from nucleotide 241 to nucleotide 1408,the other primer of said primers recognizing a sequence within theforeign DNA of said first event comprising the nucleotide sequence ofthe complement of SEQ ID No 2 from nucleotide 1452 to nucleotide 1843 orthe nucleotide sequence of SEQ ID No 3 from nucleotide 1 to nucleotide240, and comprising amplifying a DNA fragment of between 50 and 1000 bp,or between 100 and 500 bp from a nucleic acid of said second eventpresent in biological samples using a polymerase chain reaction with atleast two primers, one of said primers recognizing the 5′ flankingregion of the second elite event specified above, said 5′ flankingregion comprising the nucleotide sequence of nucleotides 1 to 209 of SEQID No 12, or the 3′ flanking region of said second elite event, said 3′flanking region comprising the nucleotide sequence of the complement ofnucleotides 569 to 1000 of SEQ ID No 13, the other primer of saidprimers recognizing a sequence within the foreign DNA of said secondevent comprising the nucleotide sequence of the complement of SEQ ID No12 from nucleotide 210 to nucleotide 720 or the nucleotide sequence ofSEQ ID No 13 from nucleotide 1 to nucleotide 568.

Also provided herein is a method for identifying a transgenic soybeanplant, or cells, parts, seed or progeny thereof comprising 2 eliteevents, wherein said plant, cells, seed, or progeny are tolerant toglyphosate, glufosinate and/or an HPPD inhibitor herbicide (such asisoxaflutole), in biological samples, said method comprising amplifyinga DNA fragment of between 50 and 1000 or between 100 and 500 bp from anucleic acid of said first event present in biological samples using apolymerase chain reaction with at least two primers, one of said primersrecognizing the 5′ flanking region of the first elite event specifiedabove, said 5′ flanking region comprising the nucleotide sequence of SEQID No 2 from nucleotide 1 to nucleotide 1451, or the 3′ flanking regionof said first elite event, said 3′ flanking region comprising thenucleotide sequence of the complement of SEQ ID No 3 from nucleotide 241to nucleotide 1408, the other primer of said primers recognizing asequence within the foreign DNA of said first event comprising thenucleotide sequence of the complement of SEQ ID No 2 from nucleotide1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No 3 fromnucleotide 1 to nucleotide 240, and comprising amplifying a DNA fragmentof between 50 and 1000 bp or between 100 and 500 bp from a nucleic acidof said second event present in biological samples using a polymerasechain reaction with at least two primers, one of said primersrecognizing the 5′ flanking region of the second elite event specifiedabove, said 5′ flanking region comprising the nucleotide sequence ofnucleotides 1 to 311 of SEQ ID No 14, or the 3′ flanking region of saidsecond elite event, said 3′ flanking region comprising the nucleotidesequence of the complement of nucleotides 508 to 1880 of SEQ ID No 15,the other primer of said primers recognizing a sequence within theforeign DNA of said second event comprising the nucleotide sequence ofthe complement of SEQ ID No 14 from nucleotide 312 to nucleotide 810 orthe nucleotide sequence of SEQ ID No 15 from nucleotide 1 to nucleotide507.

Also provided herein is a kit for identifying a transgenic soybeanplant, or cells, parts, seed or progeny thereof comprising 2 eliteevents and being tolerant to glyphosate, glufosinate and an HPPDinhibitor herbicide (such as isoxaflutole), in biological samples, saidkit comprising one primer recognizing the 5′ flanking region of thefirst elite event specified above, said 5′ flanking region comprisingthe nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide1451, or one primer recognizing the 3′ flanking region of said firstelite event, said 3′ flanking region comprising the nucleotide sequenceof the complement of SEQ ID No 3 from nucleotide 241 to nucleotide 1408,and one primer recognizing a sequence within the foreign DNA of saidfirst event, said foreign DNA comprising the nucleotide sequence of thecomplement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 orthe nucleotide sequence of SEQ ID No 3 from nucleotide 1 to nucleotide240, and said kit comprising one primer recognizing the 5′ flankingregion of the second elite event specified above, said 5′ flankingregion comprising the nucleotide sequence of nucleotides 1 to 209 of SEQID No 12, or one primer recognizing the 3′ flanking region of saidsecond elite event, said 3′ flanking region comprising the nucleotidesequence of the complement of nucleotides 569 to 1000 of SEQ ID No 13,the other primer of said primers recognizing a sequence within theforeign DNA of said second event comprising the nucleotide sequence ofthe complement of SEQ ID No 12 from nucleotide 210 to nucleotide 720 orthe nucleotide sequence of SEQ ID No 13 from nucleotide 1 to nucleotide568.

Also provided herein is a kit for identifying a transgenic soybeanplant, or cells, parts, seed or progeny thereof comprising 2 eliteevents and being tolerant to glyphosate, glufosinate and an HPPDinhibitor herbicide (such as isoxaflutole), in biological samples, saidkit comprising one primer recognizing the 5′ flanking region of thefirst elite event specified above, said 5′ flanking region comprisingthe nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide1451, or one primer recognizing the 3′ flanking region of said firstelite event, said 3′ flanking region comprising the nucleotide sequenceof the complement of SEQ ID No 3 from nucleotide 241 to nucleotide 1408,and one primer recognizing a sequence within the foreign DNA of saidfirst event, said foreign DNA comprising the nucleotide sequence of thecomplement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 orthe nucleotide sequence of SEQ ID No 3 from nucleotide 1 to nucleotide240, and said kit comprising one primer recognizing the 5′ flankingregion of the second elite event specified above, said 5′ flankingregion comprising the nucleotide sequence of nucleotides 1 to 311 of SEQID No 14, or the 3′ flanking region of said second elite event, said 3′flanking region comprising the nucleotide sequence of the complement ofnucleotides 508 to 1880 of SEQ ID No 15, the other primer of saidprimers recognizing a sequence within the foreign DNA of said secondevent comprising the nucleotide sequence of the complement of SEQ ID No14 from nucleotide 312 to nucleotide 810 or the nucleotide sequence ofSEQ ID No 15 from nucleotide 1 to nucleotide 507.

Also provided herein is a soybean plant, plant cell, tissue, or seed,comprising in their genome a nucleic acid molecule comprising anucleotide sequence with at least 97, 98, or at least 99% or 99.5%sequence identity to the nucleotide sequence of SEQ ID No. 20 fromnucleotide position 1452 to nucleotide position 16638, the nucleotidesequence of SEQ ID No. 20 from nucleotide position 2257 to nucleotideposition 16601 or their complement, or a nucleotide sequence with atleast 97, 98, or at least 99% or 99.5% sequence identity to SEQ ID No.20 or the complement thereof, and comprising in their genome a nucleicacid molecule comprising a nucleotide sequence with at least 97, 98, orat least 99% or 99.5% sequence identity to the nucleotide sequence ofEE-GM1 or EE-GM2 or the complement thereof, reference seed comprisingEE-GM1 having been deposited under deposit number NCIMB 41658, andreference seed comprising EE-GM2 having been deposited under depositnumber NCIMB 41660. In one embodiment of this invention, the nucleotidesequence of EE-GM1 or EE-GM2 in said plant, plant cell, tissue, or seed,is the DNA sequence (such as the foreign DNA sequence) in SEQ ID No 12or 13, or the DNA sequence (such as the foreign DNA sequence) in SEQ IDNo 14 or 15, or the DNA sequence in the plant genome (such as in thedeposited seeds comprising EE-GM1 or EE-GM2 of the invention) comprisingSEQ ID No 12 and 13 between the first nucleotide of the sequence of SEQID No 12 and the last nucleotide of the sequence of SEQ ID No 13, or theDNA sequence in the plant genome comprising SEQ ID No 14 and 15 betweenthe first nucleotide of the sequence of SEQ ID No 14 and the lastnucleotide of the sequence of SEQ ID No 15.

One embodiment of this invention provides a soybean plant, plant cell,tissue, or seed, comprising in their genome a nucleic acid moleculehybridizing to the nucleotide sequence of SEQ ID No 1 or the complementthereof, or hybridizing to the nucleotide sequence of SEQ ID No. 20 fromnucleotide position 1452 to nucleotide position 16638 or the complementthereof, or hybridizing to the nucleotide sequence of SEQ ID No. 20 orthe complement thereof, and comprising in their genome a nucleic acidmolecule hybridizing to the nucleotide sequence of EE-GM1 or EE-GM2 orthe complement thereof, reference seed comprising EE-GM1 having beendeposited under deposit number NCIMB 41658, and reference seedcomprising EE-GM2 having been deposited under deposit number NCIMB41660. In one embodiment of this invention, the nucleotide sequence ofEE-GM1 or EE-GM2 in said plant, plant cell, tissue, or seed, is theforeign DNA in SEQ ID No 12 or 13, or the foreign DNA in SEQ ID No 14 or15, or the foreign DNA sequence in the plant genome (such as in thedeposited seeds comprising EE-GM1 or EE-GM2 of the invention) comprisingSEQ ID No 12 and 13 between the first nucleotide of the sequence of SEQID No 12 and the last nucleotide of the sequence of SEQ ID No 13, or theforeign DNA sequence in the plant genome comprising SEQ ID No 14 and 15between the first nucleotide of the sequence of SEQ ID No 14 and thelast nucleotide of the sequence of SEQ ID No 15.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Examples, not intended to limit the invention to specificembodiments described, may be understood in conjunction with theaccompanying Figures, incorporated herein by reference, in which:

FIG. 1: Schematic representation of the relationship between the citednucleotide sequences and primers for elite event EE-GM3. black bar:foreign DNA; hatched bar: DNA of plant origin; checkered arrow (a):chimeric HPPD PF W366 encoding gene (see Table 1 for composition of thechimeric gene); hatched arrow (b): chimeric 2mEPSPS encoding gene (seeTable 1 for composition of the chimeric gene); black arrows:oligonucleotide primers, the figures under the bars represent nucleotidepositions; (c) refers to complement of the indicated nucleotidesequence; Note: the scheme is not drawn to scale.

FIG. 2: Results obtained by the PCR Identification Protocol developedfor EE-GM3. Loading sequence of the gel: Lane 1: Molecular weight marker(100 bp ladder); lanes 2 and 3: DNA samples from soybean plantscomprising the transgenic event EE-GM3; lanes 4-7: DNA samples fromtransgenic soybean plants not comprising elite event EE-GM3, butcomprising the same herbicide tolerance genes (other transformationevents); lane 8: DNA sample from wt soybean; lane 9: no template DNAcontrol; lane 10: molecular weight marker.

FIG. 3: Schematic representation of the relationship between the citednucleotide sequences and primers for elite event EE-GM1. black bar:foreign DNA; hatched bar: DNA of plant origin; horizontally stripedarrow (d): chimeric phosphinothricin acetyltransferase encoding gene(see SEQ ID No. 11 for composition of the chimeric gene; black arrows:oligonucleotide primers, the figures under the bars represent nucleotidepositions; (c) refers to complement of the indicated nucleotidesequence; Note: the scheme is not drawn to scale.

FIG. 4: Schematic representation of the relationship between the citednucleotide sequences and primers for elite event EE-GM2. black bar:foreign DNA; hatched bar: DNA of plant origin; horizontally stripedarrow (d): chimeric phosphinothricin acetyltransferase encoding gene(see SEQ ID No. 11 for composition of the chimeric gene; black arrows:oligonucleotide primers, the figures under the bars represent nucleotidepositions; (c) refers to complement of the indicated nucleotidesequence; NA: not applicable. Note: the scheme is not drawn to scale.

FIG. 5: PCR Identification Protocol developed for EE-GM1. Loadingsequence of the gel: Lane 1: DNA sample from soybean plants comprisingthe transgenic event EE-GM1; lane 2: DNA sample from a transgenicsoybean plant not comprising elite event EE-GM1; lane 3: control DNAsamples from wild-type soybean plants; lane 4: no template control; lane5: molecular weight marker.

FIG. 6: PCR Identification Protocol developed for EE-GM2. Loadingsequence of the gel: Lane 1: DNA sample from soybean plants comprisingthe transgenic event EE-GM2; lane 2: DNA sample from a transgenicsoybean plant not comprising elite event EE-GM2; lane 3: control DNAsamples from wild-type soybean plants; lane 4: no template control; lane5: molecular weight marker.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The incorporation of a recombinant DNA molecule in the plant genometypically results from transformation of a cell or tissue. Theparticular site of incorporation is usually due to random integration.

The DNA introduced into the plant genome as a result of transformationof a plant cell or tissue with a recombinant DNA or “transforming DNA”,and originating from such transforming DNA is hereinafter referred to as“foreign DNA” comprising one or more “transgenes”. The transgenes ofEE-GM3 are the glyphosate and HPPD inhibitor herbicide tolerance genes.“Plant DNA” in the context of the present invention will refer to DNAoriginating from the plant which is transformed. Plant DNA will usuallybe found in the same genetic locus in the corresponding wild-type plant.The foreign DNA can be characterized by the location and theconfiguration at the site of incorporation of the recombinant DNAmolecule in the plant genome. The site in the plant genome where arecombinant DNA has been inserted is also referred to as the “insertionsite” or “target site”. Insertion of the recombinant DNA into the regionof the plant genome referred to as “pre-insertion plant DNA” can beassociated with a deletion of plant DNA, referred to as “target sitedeletion”. A “flanking region” or “flanking sequence” as used hereinrefers to a sequence of at least 20 bp, preferably at least 50 bp, andup to 5000 bp of DNA different from the introduced DNA, preferably DNAfrom the plant genome which is located either immediately upstream ofand contiguous with or immediately downstream of and contiguous with theforeign DNA. Transformation procedures leading to random integration ofthe foreign DNA will result in transformants with different flankingregions, which are characteristic and unique for each transformant. Whenthe recombinant DNA is introduced into a plant through traditionalcrossing, its insertion site in the plant genome, or its flankingregions will generally not be changed.

An “isolated nucleic acid (sequence)” or “isolated DNA (sequence)”, asused herein, refers to a nucleic acid or DNA (sequence) which is nolonger in the natural environment it was isolated from, e.g., thenucleic acid sequence in another bacterial host or in a plant genome, ora nucleic acid or DNA fused to DNA or nucleic acid from another origin,such as when contained in a chimeric gene under the control of aplant-expressible promoter.

An event is defined as a (artificial) genetic locus that, as a result ofgenetic engineering, carries a foreign DNA or transgene comprising atleast one copy of a gene of interest or of the genes of interest. Thetypical allelic states of an event are the presence or absence of theforeign DNA.

An event is characterized phenotypically by the expression of thetransgene. At the genetic level, an event is part of the genetic make-upof a plant. At the molecular level, an event can be characterized by therestriction map (e.g., as determined by Southern blotting), by theupstream and/or downstream flanking sequences of the transgene, thelocation of molecular markers and/or the molecular configuration of thetransgene. Usually transformation of a plant with a transforming DNAcomprising at least one gene of interest leads to a population oftransformants comprising a multitude of separate events, each of whichis unique. An event is characterized by the foreign DNA and at least oneof the flanking sequences.

An elite event, as used herein, is an event which is selected from agroup of events, obtained by transformation with the same transformingDNA, based on the expression and stability of the transgene(s) and itscompatibility with optimal agronomic characteristics of the plantcomprising it. Thus the criteria for elite event selection are one ormore, preferably two or more, advantageously all of the following:

-   -   a) that the presence of the foreign DNA does not compromise        other desired characteristics of the plant, such as those        relating to agronomic performance or commercial value;    -   b) that the event is characterized by a well defined molecular        configuration which is stably inherited and for which        appropriate tools for identity control can be developed;    -   c) that the gene(s) of interest show(s) a correct, appropriate        and stable spatial and temporal phenotypic expression, both in        heterozygous (or hemizygous) and homozygous condition of the        event, at a commercially acceptable level in a range of        environmental conditions in which the plants carrying the event        are likely to be exposed in normal agronomic use.

It is preferred that the foreign DNA is associated with a position inthe plant genome that allows easy introgression into desired commercialgenetic backgrounds.

The status of an event as an elite event is confirmed by introgressionof the elite event in different relevant genetic backgrounds andobserving compliance with one, two or all of the criteria e.g. a), b)and c) above.

An “elite event” thus refers to a genetic locus comprising a foreignDNA, which meets the above-described criteria. A plant, plant materialor progeny such as seeds can comprise one or more elite events in itsgenome.

The tools developed to identify an elite event or the plant or plantmaterial comprising an elite event, or products which comprise plantmaterial comprising the elite event, are based on the specific genomiccharacteristics of the elite event, such as, a specific restriction mapof the genomic region comprising the foreign DNA, molecular markers orthe sequence of the flanking region(s) of the foreign DNA.

Once one or both of the flanking regions of the foreign DNA have beensequenced, primers and probes can be developed which specificallyrecognize this (these) sequence(s) in the nucleic acid (DNA or RNA) of asample by way of a molecular biological technique. For instance a PCRmethod can be developed to identify the elite event in biologicalsamples (such as samples of plants, plant material or productscomprising plant material). Such a PCR is based on at least two specific“primers”, one recognizing a sequence within the 5′ or 3′ flankingregion of the elite event and the other recognizing a sequence withinthe foreign DNA. The primers preferably have a sequence of between 15and 35 nucleotides which under optimized PCR conditions “specificallyrecognize” a sequence within the 5′ or 3′ flanking region of the eliteevent and the foreign DNA of the elite event respectively, so that aspecific fragment (“integration fragment” or discriminating amplicon) isamplified from a nucleic acid sample comprising the elite event. Thismeans that only the targeted integration fragment, and no other sequencein the plant genome or foreign DNA, is amplified under optimized PCRconditions.

PCR primers suitable for identification of EE-GM3 may be the following:

-   -   oligonucleotides ranging in length from 17 nt to about 200 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 consecutive nucleotides, selected        from the plant DNA in the 5′ flanking sequence (SEQ ID No 2 from        nucleotide 1 to nucleotide 1451) at their 3′ end (primers        recognizing 5′ flanking sequences); or    -   oligonucleotides ranging in length from 17 nt to about 200 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 consecutive nucleotides, selected        from the plant DNA in the 3′ flanking sequence (complement of        SEQ ID No 3 from nucleotide 241 to nucleotide 1408) at their 3′        end (primers recognizing 3′ flanking sequences); or    -   oligonucleotides ranging in length from 17 nt to about 200 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 consecutive nucleotides, selected        from the inserted DNA sequences (complement of SEQ ID No 2 from        nucleotide 1452 to nucleotide 1843) at their 3′ end (primers        recognizing foreign DNA); or    -   oligonucleotides ranging in length from 17 nt to about 200 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 consecutive nucleotides, selected        from the inserted DNA sequences (SEQ ID No 3 from nucleotide 1        to nucleotide 240); or    -   suitable oligonucleotides ranging in length from 17 nt to about        200 nt, comprising a nucleotide sequence of at least 17        consecutive nucleotides, preferably 20 consecutive nucleotides,        selected from the nucleotide sequence of the inserted DNA        fragment or its complement (SEQ ID No 1 or SEQ ID No 20 from        nucleotide position 1452 to 16638).

The primers may of course be longer than the mentioned 17 consecutivenucleotides, and may, e.g., be 20, 21, 30, 35, 50, 75, 100, 150, 200 ntlong or even longer. The primers may entirely consist of nucleotidesequence selected from the mentioned nucleotide sequences of flankingsequences and foreign DNA sequences. However, the nucleotide sequence ofthe primers at their 5′ end (i.e. outside of the 3′-located 17consecutive nucleotides) is less critical. Thus, the 5′ sequence of theprimers may comprise or consist of a nucleotide sequence selected fromthe flanking sequences or foreign DNA, as appropriate, but may containseveral (e.g., 1, 2, 5, or 10) mismatches. The 5′ sequence of theprimers may even entirely be a nucleotide sequence unrelated to theflanking sequences or foreign DNA, such as, e.g., a nucleotide sequencerepresenting one or more restriction enzyme recognition sites. Suchunrelated sequences or flanking DNA sequences with mismatches shouldpreferably be not longer than 100, more preferably not longer than 50 oreven 25 nucleotides.

Moreover, suitable primers may comprise, consist or consist essentiallyof a nucleotide sequence at their 3′ end spanning the joining regionbetween the plant DNA derived sequences and the foreign DNA sequences(located at nucleotides 1451-1452 in SEQ ID No 2 and nucleotides 240-241in SEQ ID No 3 for EE-GM3) provided the mentioned 3′-located 17consecutive nucleotides are not derived exclusively from either theforeign DNA or plant-derived sequences in SEQ ID No 2 or 3.

It will also be immediately clear to the skilled artisan that properlyselected PCR primer pairs should also not comprise sequencescomplementary to each other.

For the purpose of the invention, the “complement of a nucleotidesequence represented in SEQ ID No: X” is the nucleotide sequence whichcan be derived from the represented nucleotide sequence by replacing thenucleotides with their complementary nucleotide according to Chargaff'srules (A

T; G

C) and reading the sequence in the 5′ to 3′ direction, i.e., in oppositedirection of the represented nucleotide sequence.

Examples of suitable primers for EE-GM3 are the oligonucleotidesequences of SEQ ID No 5 (3′ flanking sequence recognizing primer), SEQID No 4 (foreign DNA recognizing primer for use with a 3′ flankingsequence recognizing primers), or SEQ ID No 7 (foreign DNA recognizingprimer for use with a 3′ flanking sequence recognizing primers).

Other examples of suitable oligonucleotide primers for EE-GM3 compriseat their 3′ end the following sequences or consist (essentially) of suchsequences:

-   a. 5′ flanking sequence recognizing primers:    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 264 to        nucleotide 283    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 266 to        nucleotide 285    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1240 to        nucleotide 1259    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 265 to        nucleotide 285    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 265 to        nucleotide 283    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1239 to        nucleotide 1259    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1241 to        nucleotide 1259    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1244 to        nucleotide 1263    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1248 to        nucleotide 1267    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1250 to        nucleotide 1269    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 262 to        nucleotide 279    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 263 to        nucleotide 279    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 264 to        nucleotide 285    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 266 to        nucleotide 283    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1238 to        nucleotide 1259    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1242 to        nucleotide 1259    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1243 to        nucleotide 1263    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1245 to        nucleotide 1263    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1247 to        nucleotide 1267    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1249 to        nucleotide 1269    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1249 to        nucleotide 1267    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 263 to        nucleotide 285    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 267 to        nucleotide 283    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1242 to        nucleotide 1263    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1243 to        nucleotide 1259    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1246 to        nucleotide 1267    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1246 to        nucleotide 1263    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1248 to        nucleotide 1269    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1250 to        nucleotide 1271    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1250 to        nucleotide 1267    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1241 to        nucleotide 1263    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1245 to        nucleotide 1267    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1247 to        nucleotide 1269    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1247 to        nucleotide 1263    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1249 to        nucleotide 1271    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1242 to        nucleotide 1261    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1241 to        nucleotide 1261    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1243 to        nucleotide 1261    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1240 to        nucleotide 1261    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1244 to        nucleotide 1261    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1239 to        nucleotide 1261    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1245 to        nucleotide 1261-   b. foreign DNA sequence recognizing primers for use with 5′ flanking    sequence recognizing primers:    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1732 to nucleotide 1751    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1735 to nucleotide 1754    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1731 to nucleotide 1750    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1732 to nucleotide 1750    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1732 to nucleotide 1752    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1731 to nucleotide 1749    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1732 to nucleotide 1749    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1731 to nucleotide 1751    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1732 to nucleotide 1753    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1731 to nucleotide 1748    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1732 to nucleotide 1748    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1735 to nucleotide 1751    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1731 to nucleotide 1752    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1732 to nucleotide 1754    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1731 to nucleotide 1747    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1731 to nucleotide 1753    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1727 to nucleotide 1746    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1727 to nucleotide 1745    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1727 to nucleotide 1747    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1727 to nucleotide 1744    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1727 to nucleotide 1748    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1727 to nucleotide 1749    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1726 to nucleotide 1745    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1726 to nucleotide 1744    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1726 to nucleotide 1746    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1726 to nucleotide 1747    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1726 to nucleotide 1748    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1724 to nucleotide 1744    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1724 to nucleotide 1745    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1724 to nucleotide 1746    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1461 to nucleotide 1478    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1670 to nucleotide 1686    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1469 to nucleotide 1486    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1508 to nucleotide 1527    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1667 to nucleotide 1686    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1670 to nucleotide 1687    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1673 to nucleotide 1689    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1688 to nucleotide 1704    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1688 to nucleotide 1705    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1692 to nucleotide 1709    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1467 to nucleotide 1486    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1481 to nucleotide 1497    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1481 to nucleotide 1498    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1491 to nucleotide 1507    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1491 to nucleotide 1508    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1672 to nucleotide 1688    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1673 to nucleotide 1690    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1673 to nucleotide 1691    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1688 to nucleotide 1706    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1691 to nucleotide 1707    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1691 to nucleotide 1708    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1469 to nucleotide 1487    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1481 to nucleotide 1499    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1489 to nucleotide 1505    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1489 to nucleotide 1506    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1489 to nucleotide 1507    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1489 to nucleotide 1508    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1666 to nucleotide 1686    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1667 to nucleotide 1687    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1670 to nucleotide 1688    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1672 to nucleotide 1689    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1688 to nucleotide 1707    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1691 to nucleotide 1709    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1692 to nucleotide 1710    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1481 to nucleotide 1500    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1491 to nucleotide 1509    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1670 to nucleotide 1689    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1672 to nucleotide 1690    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1672 to nucleotide 1691    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1687 to nucleotide 1705    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1687 to nucleotide 1706    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1691 to nucleotide 1710    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1472 to nucleotide 1488    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1488 to nucleotide 1507    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1491 to nucleotide 1510    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1495 to nucleotide 1512    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1495 to nucleotide 1513    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1495 to nucleotide 1514    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1673 to nucleotide 1692    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1678 to nucleotide 1694    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1678 to nucleotide 1695    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1678 to nucleotide 1696    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1687 to nucleotide 1703    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1687 to nucleotide 1704    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1692 to nucleotide 1711    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1469 to nucleotide 1488    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1488 to nucleotide 1506    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1491 to nucleotide 1511    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1670 to nucleotide 1690    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1678 to nucleotide 1697    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1688 to nucleotide 1709    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1467 to nucleotide 1487    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1488 to nucleotide 1508    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1495 to nucleotide 1511    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1491 to nucleotide 1512    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1666 to nucleotide 1687    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1667 to nucleotide 1688    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1672 to nucleotide 1692    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1673 to nucleotide 1693    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1687 to nucleotide 1707    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1472 to nucleotide 1490    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1472 to nucleotide 1491    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1481 to nucleotide 1501    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1489 to nucleotide 1509    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1495 to nucleotide 1515    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1670 to nucleotide 1691    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1673 to nucleotide 1694    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1678 to nucleotide 1698    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1469 to nucleotide 1489    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1667 to nucleotide 1689    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1687 to nucleotide 1708    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1688 to nucleotide 1710    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1691 to nucleotide 1711    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1472 to nucleotide 1492    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1489 to nucleotide 1510    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1666 to nucleotide 1688    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1687 to nucleotide 1709    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1692 to nucleotide 1712    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1467 to nucleotide 1488    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1469 to nucleotide 1490    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1488 to nucleotide 1509    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1489 to nucleotide 1511    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1678 to nucleotide 1699    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1472 to nucleotide 1493    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1472 to nucleotide 1494    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1481 to nucleotide 1502    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1670 to nucleotide 1692    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1469 to nucleotide 1491    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1488 to nucleotide 1510    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1691 to nucleotide 1712    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1692 to nucleotide 1713    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1692 to nucleotide 1714    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1467 to nucleotide 1489    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1678 to nucleotide 1700    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1481 to nucleotide 1503    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 1691 to nucleotide 1713-   c. 3′ flanking sequence recognizing primers:    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 828 to nucleotide 847    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 830 to nucleotide 849    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 828 to nucleotide 846    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 828 to nucleotide 848    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 830 to nucleotide 848    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 830 to nucleotide 850    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 828 to nucleotide 845    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 830 to nucleotide 847    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 828 to nucleotide 849    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 830 to nucleotide 851    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 828 to nucleotide 844    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 830 to nucleotide 846    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 828 to nucleotide 850    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 830 to nucleotide 852    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 992 to nucleotide 1009    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 731 to nucleotide 752    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 776 to nucleotide 795    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 731 to nucleotide 753    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 776 to nucleotide 794    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 776 to nucleotide 796    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 776 to nucleotide 793    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 776 to nucleotide 797    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 776 to nucleotide 792    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 776 to nucleotide 798    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 733 to nucleotide 752    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 733 to nucleotide 753    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 733 to nucleotide 754    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 733 to nucleotide 755    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 838 to nucleotide 854    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 246 to nucleotide 263    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 838 to nucleotide 855    -   the complement of the nucleotide sequence of SEQ ID No 3 from        nucleotide 245 to nucleotide 264-   d. foreign DNA sequence recognizing primers for use with 3′ flanking    sequence recognizing primers:    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 173 to        nucleotide 192    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 22 to        nucleotide 41    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 172 to        nucleotide 192    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 174 to        nucleotide 192    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 191 to        nucleotide 210    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 171 to        nucleotide 192    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 175 to        nucleotide 192    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 190 to        nucleotide 210    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 192 to        nucleotide 210    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 176 to        nucleotide 192    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 189 to        nucleotide 210    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 193 to        nucleotide 210    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 188 to        nucleotide 210    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 194 to        nucleotide 210    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 199 to        nucleotide 218    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 200 to        nucleotide 218    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 197 to        nucleotide 218    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 201 to        nucleotide 218    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 201 to        nucleotide 220    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 200 to        nucleotide 220    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 199 to        nucleotide 220    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 200 to        nucleotide 221    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 199 to        nucleotide 221    -   the nucleotide sequence of SEQ ID No 3 from nucleotide 150 to        nucleotide 172

PCR primers suitable for the identification of EE-GM1 have beendescribed in WO2006/108674, particularly on page 8, line 4 to page 26,line 7 (herein incorporated by reference).

PCR primers suitable for the identification of EE-GM2 have beendescribed in WO2006/108675, particularly on pages 8, line 4 to page 33,line 4 (herein incorporated by reference).

As used herein, “the nucleotide sequence of SEQ ID No. Z from position Xto position Y” indicates the nucleotide sequence including bothnucleotide endpoints.

Preferably, the amplified fragment has a length of between 50 and 500nucleotides, such as a length between 100 and 350 nucleotides. Thespecific primers may have a sequence which is between 80 and 100%identical to a sequence within the 5′ or 3′ flanking region of the eliteevent and the foreign DNA of the elite event, respectively, provided themismatches still allow specific identification of the elite event withthese primers under optimized PCR conditions. The range of allowablemismatches however, can easily be determined experimentally and areknown to a person skilled in the art.

Detection of integration fragments can occur in various ways, e.g., viasize estimation after gel analysis. The integration fragments may alsobe directly sequenced. Other sequence specific methods for detection ofamplified DNA fragments are also known in the art.

As the sequence of the primers and their relative location in the genomeare unique for the elite event, amplification of the integrationfragment will occur only in biological samples comprising (the nucleicacid of) the elite event. Preferably when performing a PCR to identifythe presence of elite events EE-GM3 and EE-GM1 or elite events EE-GM3and EE-GM2 in unknown samples, a control is included of a set of primerswith which a fragment within a “housekeeping gene” of the plant speciesof the event can be amplified. Housekeeping genes are genes that areexpressed in most cell types and which are concerned with basicmetabolic activities common to all cells. Preferably, the fragmentamplified from the housekeeping gene is a fragment which is larger thanthe amplified integration fragment. Depending on the samples to beanalyzed, other controls can be included.

Standard PCR protocols are described in the art, such as in ‘PCRApplications Manual (Roche Molecular Biochemicals, 2nd Edition, 1999)and other references. The optimal conditions for the PCR, including thesequence of the specific primers, are specified in a “PCR (or PolymeraseChain Reaction) Identification Protocol” for each elite event. It ishowever understood that a number of parameters in the PCR IdentificationProtocol may need to be adjusted to specific laboratory conditions, andmay be modified slightly to obtain similar results. For instance, use ofa different method for preparation of DNA may require adjustment of, forinstance, the amount of primers, polymerase and annealing conditionsused. Similarly, the selection of other primers may dictate otheroptimal conditions for the PCR Identification Protocol. Theseadjustments will however be apparent to a person skilled in the art, andare furthermore detailed in current PCR application manuals such as theone cited above.

Alternatively, specific primers can be used to amplify integrationfragments that can be used as “specific probes” for identifying EE-GM3and EE-GM1 or EE-GM3 and EE-GM2 in biological samples. Contactingnucleic acid of a biological sample, with the probes, under conditionswhich allow hybridization of the probes with their correspondingfragments in the sample nucleic acid, results in the formation ofnucleic acid/probe hybrids. The formation of these hybrids can bedetected (e.g. via labeling of the nucleic acid or probe), whereby theformation of these hybrids indicates the presence of EE-GM3 and EE-GM1or EE-GM3 and EE-GM2. Such identification methods based on hybridizationwith a specific probe (either on a solid phase carrier or in solution)have been described in the art. The specific probe is preferably asequence which, under optimized conditions, hybridizes specifically to aregion within the 5′ or 3′ flanking region of the elite event andpreferably also comprising part of the foreign DNA contiguous therewith(hereinafter referred to as “specific region”). Preferably, the specificprobe comprises a sequence of between 50 and 500 bp, preferably of 100to 350 bp which is at least 80%, preferably between 80 and 85%, morepreferably between 85 and 90%, especially preferably between 90 and 95%,most preferably between 95% and 100% identical (or complementary) to thenucleotide sequence of a specific region. Preferably, the specific probewill comprise a sequence of about 15 to about 100 contiguous nucleotidesidentical (or complementary) to a specific region of the elite event.

Furthermore, detection methods specific for elite events EE-GM3 andEE-GM1 or for elite events EE-GM3 and EE-GM2 which differ from PCR basedamplification methods can also be developed using the elite eventspecific sequence information provided herein. Such alternativedetection methods include linear signal amplification detection methodsbased on invasive cleavage of particular nucleic acid structures, alsoknown as Invader™ technology, (as described e.g. in U.S. Pat. No.5,985,557 “Invasive Cleavage of Nucleic Acids”, 6,001,567 Detection ofNucleic Acid sequences by Invader Directed Cleavage, incorporated hereinby reference). For EE-GM3 detection by this method, the target sequencemay hybridized with a labeled first nucleic acid oligonucleotidecomprising the nucleotide sequence of SEQ ID No 2 from nucleotide 1452to nucleotide 1469 or its complement or said labeled nucleic acid probecomprising the nucleotide sequence of SEQ ID No 3 from nucleotide 223 tonucleotide 240 or its complement and is further hybridized with a secondnucleic acid oligonucleotide comprising the nucleotide sequence of SEQID No 2 from nucleotide 1434 to nucleotide 1451 or its complement orsaid labeled nucleic acid probe comprising the nucleotide sequence ofSEQ ID No 3 from nucleotide 241 to nucleotide 258 or its complement,wherein the first and second oligonucleotide overlap by at least onenucleotide. The duplex or triplex structure which is produced by thishybridization allows selective probe cleavage with an enzyme (Cleavase®)leaving the target sequence intact. The cleaved labeled probe issubsequently detected, potentially via an intermediate step resulting infurther signal amplification. For EE-GM1 detection by this method, thetarget sequence may hybridized with a labeled first nucleic acidoligonucleotide comprising the nucleotide sequence of SEQ ID No 12 fromnucleotide 210 to nucleotide 227 or its complement or said labelednucleic acid probe comprising the nucleotide sequence of SEQ ID No 13from nucleotide 561 to nucleotide 568 or its complement and is furtherhybridized with a second nucleic acid oligonucleotide comprising thenucleotide sequence of SEQ ID No 12 from nucleotide 192 to nucleotide209 or its complement or said labeled nucleic acid probe comprising thenucleotide sequence of SEQ ID No 13 from nucleotide 569 to nucleotide586 or its complement, wherein the first and second oligonucleotideoverlap by at least one nucleotide. The duplex or triplex structurewhich is produced by this hybridization allows selective probe cleavagewith an enzyme (Cleavase®) leaving the target sequence intact. Thecleaved labeled probe is subsequently detected, potentially via anintermediate step resulting in further signal amplification. For EE-GM2detection by this method, the target sequence may hybridized with alabeled first nucleic acid oligonucleotide comprising the nucleotidesequence of SEQ ID No 14 from nucleotide 312 to nucleotide 329 or itscomplement or said labeled nucleic acid probe comprising the nucleotidesequence of SEQ ID No 15 from nucleotide 490 to nucleotide 507 or itscomplement and is further hybridized with a second nucleic acidoligonucleotide comprising the nucleotide sequence of SEQ ID No 14 fromnucleotide 294 to nucleotide 311 or its complement or said labelednucleic acid probe comprising the nucleotide sequence of SEQ ID No 15from nucleotide 508 to nucleotide 525 or its complement, wherein thefirst and second oligonucleotide overlap by at least one nucleotide. Theduplex or triplex structure which is produced by this hybridizationallows selective probe cleavage with an enzyme (Cleavase®) leaving thetarget sequence intact. The cleaved labeled probe is subsequentlydetected, potentially via an intermediate step resulting in furthersignal amplification.

A “kit” as used herein refers to a set of reagents for the purpose ofperforming the method of the invention, more particularly, theidentification of the elite event EE-GM3 in biological samples or thedetermination of the zygosity status of EE-GM3 containing plantmaterial. More particularly, a preferred embodiment of the kit of theinvention comprises at least one or two specific primers, as describedabove for identification of the elite event, or three specific primersfor the determination of the zygosity status. Optionally, the kit canfurther comprise any other reagent described herein in the PCRIdentification Protocol. Alternatively, according to another embodimentof this invention, the kit can comprise a specific probe, as describedabove, which specifically hybridizes with nucleic acid of biologicalsamples to identify the presence of EE-GM3 therein. Optionally, the kitcan further comprise any other reagent (such as but not limited tohybridizing buffer, label) for identification of EE-GM3 in biologicalsamples, using the specific probe.

The kit of the invention can be used, and its components can bespecifically adjusted, for purposes of quality control (e.g., purity ofseed lots), detection of the presence or absence of the elite event inplant material or material comprising or derived from plant material,such as but not limited to food or feed products.

As used herein, “sequence identity” with regard to nucleotide sequences(DNA or RNA), refers to the number of positions with identicalnucleotides divided by the number of nucleotides in the shorter of thetwo sequences. The alignment of the two nucleotide sequences isperformed by the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983,Proc. Nat. Acad. Sci. USA 80:726) using a window-size of 20 nucleotides,a word length of 4 nucleotides, and a gap penalty of 4.Computer-assisted analysis and interpretation of sequence data,including sequence alignment as described above, can, e.g., beconveniently performed using the sequence analysis software package ofthe Genetics Computer Group (GCG, University of Wisconsin BiotechnologyCenter). Sequences are indicated as “essentially similar” when suchsequences have a sequence identity of at least about 75%, particularlyat least about 80%, more particularly at least about 85%, quiteparticularly at least about 90%, especially at least about 95%, moreespecially at least about 98%, or at least about 99%. It is clear thatwhen RNA sequences are said to be essentially similar or have a certaindegree of sequence identity with DNA sequences, thymidine (T) in the DNAsequence is considered equal to uracil (U) in the RNA sequence. Also, itis clear that small differences or mutations may appear in DNA sequencesover time and that some mismatches can be allowed for the event-specificprimers or probes of the invention, so any DNA sequence indicated hereinin any embodiment of this invention for any 3′ or 5′ flanking DNA or forany insert or foreign DNA or any primer or probe of this invention, alsoincludes sequences essentially similar to the sequences provided herein,such as sequences hybridizing to or with at least 90%, 95%, 96%, 97%,98%, or at least 99% sequence identity to the sequence given for any 3′or 5′ flanking DNA, for any primer or probe or for any insert or foreignDNA of this invention.

The term “primer” as used herein encompasses any nucleic acid that iscapable of priming the synthesis of a nascent nucleic acid in atemplate-dependent process, such as PCR. Typically, primers areoligonucleotides from 10 to 30 nucleotides, but longer sequences can beemployed. Primers may be provided in double-stranded form, though thesingle-stranded form is preferred. Probes can be used as primers, butare designed to bind to the target DNA or RNA and need not be used in anamplification process.

The term “recognizing” as used herein when referring to specificprimers, refers to the fact that the specific primers specificallyhybridize to a nucleic acid sequence in the elite event under theconditions set forth in the method (such as the conditions of the PCRIdentification Protocol), whereby the specificity is determined by thepresence of positive and negative controls.

The term “hybridizing” as used herein when referring to specific probes,refers to the fact that the probe binds to a specific region in thenucleic acid sequence of the elite event under standard stringencyconditions. Standard stringency conditions as used herein refers to theconditions for hybridization described herein or to the conventionalhybridizing conditions as described by Sambrook et al., 1989 (MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press, NY) which for instance can comprise the followingsteps: 1) immobilizing plant genomic DNA fragments on a filter, 2)prehybridizing the filter for 1 to 2 hours at 42° C. in 50% formamide,5×SSPE, 2×Denhardt's reagent and 0.1% SDS, or for 1 to 2 hours at 68° C.in 6×SSC, 2×Denhardt's reagent and 0.1% SDS, 3) adding the hybridizationprobe which has been labeled, 4) incubating for 16 to 24 hours, 5)washing the filter for 20 min. at room temperature in 1×SSC, 0.1% SDS,6) washing the filter three times for 20 min. each at 68° C. in 0.2×SSC,0.1% SDS, and 7) exposing the filter for 24 to 48 hours to X-ray film at−70° C. with an intensifying screen.

As used in herein, a biological sample is a sample of a plant, plantmaterial or products comprising plant material. The term “plant” isintended to encompass soybean (Glycine max) plant tissues, at any stageof maturity, as well as any cells, tissues, or organs taken from orderived from any such plant, including without limitation, any seeds,leaves, stems, flowers, roots, single cells, gametes, cell cultures,tissue cultures or protoplasts. “Plant material”, as used herein refersto material which is obtained or derived from a plant. Productscomprising plant material relate to food, feed or other products whichare produced using plant material or can be contaminated by plantmaterial. It is understood that, in the context of the presentinvention, such biological samples are tested for the presence ofnucleic acids specific for EE-GM3, EE-GM1 and EE-GM2 implying thepresence of nucleic acids in the samples. Thus the methods referred toherein for identifying elite event EE-GM3 and EE-GM2 or EE-GM3 andEE-GM1 in biological samples, relate to the identification in biologicalsamples of nucleic acids which comprise the elite event.

As used herein “comprising” is to be interpreted as specifying thepresence of the stated features, integers, steps, reagents or componentsas referred to, but does not preclude the presence or addition of one ormore features, integers, steps or components, or groups thereof. Thus,e.g., a nucleic acid or protein comprising a sequence of nucleotides oramino acids, may comprise more nucleotides or amino acids than theactually cited ones, i.e., be embedded in a larger nucleic acid orprotein. A chimeric gene comprising a DNA sequence which is functionallyor structurally defined, may comprise additional DNA sequences, such aspromoter and transcript termination sequences.

The present invention also relates to the development of a stack ofelite event EE-GM3 and elite event EE-GM1 or a stack of elite eventEE-GM3 and elite event EE-GM2 in soybean, to the plants comprising astack of these events, the progeny obtained from these plants and to theplant cells, or plant material derived from plants comprising thesestacks. Plants comprising elite event EE-GM3 and EE-GM1 or EE-GM3 andEE-GM2 can be obtained as described in example 1. Stacks are obtained bycrossing plants comprising single events using conventional breedingmethods and identification of progeny thereof comprising two differentevents.

Soybean plants or plant material comprising EE-GM3 and EE-GM1 can beidentified according to the PCR Identification Protocol described forEE-GM3 and EE-GM1 in Example 2. Briefly, soybean genomic DNA present inthe biological sample is amplified by PCR using a primer whichspecifically recognizes a sequence within the 5′ or 3′ flanking sequenceof EE-GM3 such as the primer with the sequence of SEQ ID NO: 5, and aprimer which recognizes a sequence in the foreign DNA, such as theprimer with the sequence of SEQ ID NO: 4 and further using a primerwhich specifically recognizes a sequence within the 5′ or 3′ flankingsequence of EE-GM1 such as the primer with the sequence of SEQ ID NO:16, and a primer which recognizes a sequence in the foreign DNA, such asthe primer with the sequence of SEQ ID NO: 17.

Soybean plants or plant material comprising EE-GM3 and EE-GM2 can beidentified according to the PCR Identification Protocol described forEE-GM3 and EE-GM2 in Example 2. Briefly, soybean genomic DNA present inthe biological sample is amplified by PCR using a primer whichspecifically recognizes a sequence within the 5′ or 3′ flanking sequenceof EE-GM3 such as the primer with the sequence of SEQ ID NO: 5, and aprimer which recognizes a sequence in the foreign DNA, such as theprimer with the sequence of SEQ ID NO: 4 and further using a primerwhich specifically recognizes a sequence within the 5′ or 3′ flankingsequence of EE-GM2 such as the primer with the sequence of SEQ ID NO:18, and a primer which recognizes a sequence in the foreign DNA, such asthe primer with the sequence of SEQ ID NO: 19.

DNA primers which amplify part of an endogenous soybean sequence areused as positive control for the PCR amplification. If upon PCRamplification, the material yields the fragments of the expected sizes,the material contains plant material from a soybean plant harboringelite event EE-GM3 and EE-GM1 or from a soybean plant harboring eliteevent EE-GM3 and EE-GM2.

Plants harboring EE-GM3 and EE-GM1 or EE-GM3 and EE-GM2 arecharacterized by their glyphosate tolerance, as well as by theirtolerance to HPPD inhibitors such as isoxaflutol and further by theirtolerance to glufosinate. Plants harboring the event stacks are alsocharacterized by having agronomical characteristics that are comparableto commercially available varieties of soybean, in the absence ofherbicide application. It has been observed that the presence of foreignDNA in the insertion regions of the soybean plant genome describedherein, confers particularly interesting phenotypic and molecularcharacteristics to the plants comprising this event.

One embodiment of this invention provides a combination or elite eventsEE-GM3 and EE-GM1 and/or EE-GM2 in soybean plants, obtainable byinsertion of transgenes at specific locations in the soybean genome,which elite events confers tolerance to glyphosate, glufosinate and anHPPD inhibitor herbicide such as isoxaflutole on such soybean plants,and wherein such elite events do not cause any effect on the agronomicperformance of such soybeans negatively affecting the yield of suchsoybean plants, compared to isogenic lines (as used herein, “isogeniclines” or “near-isogenic lines” are soybean lines of the same geneticbackground but lacking the transgenes, such as plants of the samegenetic background as the plant used for transformation, or segregatingsister lines having lost the transgenes). Particularly, the currentinvention provides a combination of elite events EE-GM3 and EE-GM1and/or EE-GM2 in soybean plants, wherein the insertion or presence ofsaid elite event in the genome of such soybean plants does not cause anincreased susceptibility to disease, does not cause a yield drag, ordoes not cause increased lodging, in such soybean plants, as compared toisogenic lines. Hence, the current invention provides a combination ofelite events in soybean plants, designated as EE-GM3 and EE-GM1 and/orEE-GM2, which results in soybean plants that can tolerate theapplication of glyphosate, glufosinate and an HPPD inhibitor herbicide(either simultaneously or separately) without negatively affecting theyield of said soybean plants compared to isogenic lines, which soybeanplants have no statistically significant difference in their diseasesusceptibility, or lodging, compared to isogenic soybean plants. Thesecharacteristics make the current combination of elite events veryinteresting to control glyphosate-resistant weeds in soybean fields, andcan also be used in approaches to prevent or delay further glyphosateresistance development in soybean fields (e.g., by application ofglyphosate and isoxaflutole and/or glufosinate, or by application ofisoxaflutole and glyphosate and/or glufosinate, or by application ofglufosinate and glyphosate and/or isoxafulote, securing at least 2 oreven 3 different modes of actions of herbicides applied on a soybeanfield).

Provided herein is also a soybean plant or part thereof comprising eventEE-GM3 and elite event EE-GM1 or EE-GM2, wherein representative soybeanseed comprising event EE-GM3 has been deposited under NCIMB accessionnumber 41659, representative soybean seed comprising elite event EE-GM1has been deposited at the NCIMB under accession number NCIMB 41658,representative seed comprising elite event EE-GM2 has been deposited atthe NCIMB under accession number NCIMB 41660, representative soybeanseed comprising event EE-GM3 and EE-GM1 has been deposited at the ATCCunder accession number PTA-11041, and representative soybean seedcomprising event EE-GM3 and EE-GM2 has been deposited at the ATCC underaccession number PTA-11042.

Soybean plants or parts thereof comprising EE-GM3 and EE-GM1 or EE-GM3and EE-GM2 may be obtained by combining the respective elite events ascan be found in the respective deposited seeds through any meansavailable in the art, including by crossing plants from the depositedseeds, collecting the progeny thereof, and identifying those progenyplants comprising the appropriate combination of elite events. Furtherprovided herein are seeds of such plants, comprising such events, aswell as a soybean product produced from such seeds, wherein said soybeanproduct comprises event EE-GM3 and EE-GM1 or EE-GM2. Such soybeanproduct can be or can comprise meal, ground seeds, flour, flakes, etc.Particularly, such soybean product comprises a nucleic acid thatproduces amplicons diagnostic for event EE-GM3 and elite event EE-GM1 orEE-GM2, such amplicons comprising SEQ ID No. 2 or 3, SEQ ID No. 14 or 15and/or SEQ ID No. 12 or 13. Also provided herein is a method forproducing a soybean product, comprising obtaining soybean seedcomprising event EE-GM3 and elite event EE-GM1 or EE-GM2, and producingsuch soybean product therefrom.

Also provided herein is a soybean plant, which is progeny of any of theabove soybean plants, and which comprises event EE-GM3 and EE-GM1 orEE-GM2.

Further provided herein is a method for producing a soybean planttolerant to glyphosate and/or glufosinate and/or isoxaflutoleherbicides, comprising introducing into the genome of such plant eventEE-GM3 and event EE-GM1 or EE-GM2, particularly by crossing a firstsoybean plant containing event EE-GM3 with a soybean plant comprisingEE-GM1 and/or EE-GM2, and selecting a progeny plant tolerant toglyphosate and/or glufosinate and/or isoxaflutole.

Also provided herein is a glyphosate and glufosinate and/or isoxaflutoletolerant plant, particularly without yield drag, and with acceptableagronomical characteristics, comprising a 2mEPSPS, HPPD and PAT protein,and capable of producing an amplicon diagnostic for events EE-GM3 andEE-GM1 or EE-GM3 and EE-GM2.

Further provided herein is a method for controlling weeds in a field ofsoybean plants comprising events EE-GM3 and EE-GM1 or EE-GM3 and EE-GM2,or a field to be planted with such soybean plants, comprising treatingthe field with an effective amount of an isoxaflutole-based,glyphosate-based and/or glufosinate-based herbicide, wherein such plantsare tolerant to such herbicide(s).

Further provided herein is a soybean plant, cell, tissue or seed,comprising EE-GM3 and EE-GM1, characterized by comprising in the genomeof its cells a nucleic acid sequence with at least 80%, 90%, 95% or 100%sequence identity to SEQ ID No. 2 from nucleotide 1431 to 1472 and anucleic acid sequence with at least 80%, 90%, 95% or 100% sequenceidentity to SEQ ID No. 3 from nucleotide 220 to 261, or the complementof said sequences, and also a nucleic acid sequence with at least 80%,90%, 95% or 100% sequence identity to SEQ ID No. 12 from nucleotide 199to 220 and a nucleic acid sequence with at least 80%, 90%, 95% or 100%sequence identity to SEQ ID No. 13 from nucleotide 558 to 579, or thecomplement of said sequences.

Also provided herein is a soybean plant, cell, tissue or seed,comprising EE-GM3 and EE-GM2, characterized by comprising in the genomeof its cells a nucleic acid sequence with at least 80%, 90%, 95% or 100%sequence identity to SEQ ID No. 2 from nucleotide 1431 to 1472 and anucleic acid sequence with at least 80%, 90%, 95% or 100% sequenceidentity to SEQ ID No. 3 from nucleotide 220 to 261, or the complementof said sequences, and also a nucleic acid sequence with at least 80%,90%, 95% or 100% sequence identity to SEQ ID No. 14 from nucleotide 301to 322 and a nucleic acid sequence with at least 80%, 90%, 95% or 100%sequence identity to SEQ ID No. 15 from nucleotide 497 to 518, or thecomplement of said sequences.

The term “isoxaflutole”, as used herein, refers to the herbicideisoxaflutole[i.e.(5-cyclopropyl-4-isoxazolyl)[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]methanone],the active metabolite thereof, diketonitrile, and any mixtures orsolutions comprising said compounds. HPPD inhibiting herbicides usefulfor application on the plants comprising the events of this inventionare the diketonitriles, e.g.2-cyano-3-cyclopropyl-1-(2-methylsulphonyl-4-trifluoromethylphenyl)-propane-1,3-dioneand2-cyano-1-[4-(methylsulphonyl)-2-trifluoromethylphenyl]-3-(1-methylcyclopropyl)propane-1,3-fione;other isoxazoles; and the pyrazolinates, e.g. topramezone [i.e.[3-(4,5-dihydro-3-isoxazolyl)-2-methyl-4-(methylsulfonyl)phenyl](5-hydroxy-1-methyl-1H-pyrazol-4-yl)methanone],and pyrasulfotole[(5-hydroxy-1,3-dimethylpyrazol-4-yl(2-mesyl-4-trifluaromethylphenyl)methanone];or pyrazofen[2-[4-(2,4-dichlorobenzoyl)-1,3-dimethylpyrazol-5-yloxy]acetophenone].

In one embodiment of this invention, a field to be planted with soybeanplants containing the EE-GM3 event, can be treated with an HPPDinhibitor herbicide, such as isoxaflutole (‘IFT’), before the soybeanplants are planted or the seeds are sown, which cleans the field ofweeds that are killed by the HPPD inhibitor, allowing for no-tillpractices, followed by planting or sowing of the soybeans in that samepre-treated field later on (burndown application using an HPPD inhibitorherbicide). The residual activity of IFT will also protect the emergingand growing soybean plants from competition by weeds in the early growthstages. Once the soybean plants have a certain size, and weeds tend tore-appear, glyphosate, or an HPPD inhibitor-glyphosate mixture, can beapplied as post-emergent herbicide over the top of the plants.

In another embodiment of this invention, a field in which seedscontaining the EE-GM3 event were sown, can be treated with an HPPDinhibitor herbicide, such as IFT, before the soybean plants emerge butafter the seeds are sown (the field can be made weed-free before sowingusing other means, typically conventional tillage practices such asploughing, chissel ploughing, or seed bed preparation), where residualactivity will keep the field free of weeds killed by the herbicide sothat the emerging and growing soybean plants have no competition byweeds (pre-emergence application of an HPPD inhibitor herbicide). Oncethe soybean plants have a certain size, and weeds tend to re-appear,glyphosate—or an HPPD inhibitor-glyphosate mixture—can be applied aspost-emergent herbicide over the top of the plants.

In another embodiment of this invention, plants containing the EE-GM3event, can be treated with an HPPD inhibitor herbicide, such as IFT,over the top of the soybean plants (that have emerged from the seedsthat were sown), which cleans the field of weeds killed by the HPPDinhibitor, which application can be together with (e.g., in a spray tankmix), followed by or preceded by a treatment with glyphosate aspost-emergent herbicide over the top of the plants (post-emergenceapplication of an HPPD inhibitor herbicide (with or withoutglyphosate)).

Also, in accordance with the current invention, soybean plants harboringEE-GM3 and EE-GM1 or EE-GM2 may be treated with the followinginsectides, herbicides or fungicides or soybean seeds harboring EE-GM3and EE-GM1 or EE-GM2 may be coated with a seed coating comprising thefollowing insectides, herbicides or fungicides:

Soybean Herbicides:

Alachlor, Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl,Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin,Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim,Isoxaflutole, Glufosinate.

Soybean Insecticides:

Lambda-cyhalothrin, Methomyl, Parathion, Thiocarb, Imidacloprid,Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran,Flubendiamide, Rynaxypyr, Cyazypyr, Spinosad, Spinotoram,Emamectin-Benzoate, Fipronil, Ethiprole, Deltamethrin, β-Cyfluthrin,gamma and lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb,beta-Cyfluthrin.

Soybean Fungicides:

Azoxystrobin, Cyproconazole, Epoxiconazole, Flutriafol, Pyraclostrobin,Tebuconazole, Trifloxystrobin, Prothioconazole, Tetraconazole.

The following examples describe the identification of elite eventsEE-GM3, EE-GM1 and EE-GM2 and of plants containing a stack of eventEE-GM3 with EE-GM1 or EE-GM3 with EE-GM2 and the development of toolsfor the specific identification of elite event EE-GM3, EE-GM1 or EE-GM2and stacks thereof in biological samples.

Unless stated otherwise in the Examples, all recombinant techniques arecarried out according to standard protocols as described in “Sambrook Jand Russell D W (eds.) (2001) Molecular Cloning: A Laboratory Manual,3rd Edition, Cold Spring Harbor Laboratory Press, New York” and in“Ausubel FA, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA andStruhl K (eds.) (2006) Current Protocols in Molecular Biology. JohnWiley & Sons, New York”.

Standard materials and references are described in “Croy RDD (ed.)(1993) Plant Molecular Biology LabFax, BIOS Scientific Publishers Ltd.,Oxford and Blackwell Scientific Publications, Oxford” and in “Brown TA,(1998) Molecular Biology LabFax, 2nd Edition, Academic Press, SanDiego”. Standard materials and methods for polymerase chain reactions(PCR) can be found in “McPherson MJ and Mo/ller SG (2000) PCR (TheBasics), BIOS Scientific Publishers Ltd., Oxford” and in “PCRApplications Manual,” 3rd Edition (2006), Roche Diagnostics GmbH,Mannheim or the roche-applied-science website.

It should be understood that a number of parameters in any lab protocolsuch as the PCR protocols in the below Examples may need to be adjustedto specific laboratory conditions, and may be modified slightly toobtain similar results. For instance, use of a different method forpreparation of DNA or the selection of other primers in a PCR method maydictate other optimal conditions for the PCR protocol. These adjustmentswill however be apparent to a person skilled in the art, and arefurthermore detailed in current PCR application manuals.

In the description and examples, reference is made to the followingsequences:

-   SEQ ID No. 1: SalI fragment nucleotide sequence of vector pSF10.-   SEQ ID No. 2: nucleotide sequence comprising the 5′ region flanking    the foreign DNA comprising the herbicide tolerance genes in EE-GM3.-   SEQ ID No. 3: nucleotide sequence comprising the 3′ region flanking    the foreign DNA comprising the herbicide tolerance genes in EE-GM3.-   SEQ ID No. 4: primer SOY028-   SEQ ID No. 5: primer SOY029-   SEQ ID No. 6: primer SMP 187-   SEQ ID No. 7: primer STV019-   SEQ ID No. 8: nucleotide sequence of the amplicon-   SEQ ID No. 9: primer 1 for amplification of control fragment (SOY01)-   SEQ ID No. 10: primer 2 for amplification of control fragment    (SOY02)-   SEQ ID No. 11: nucleotide sequence of pB2/P35SAcK-   SEQ ID No. 12: nucleotide sequence comprising the 5′ region flanking    the foreign DNA in EE-GM1-   SEQ ID No. 13: nucleotide sequence comprising the 3′ region flanking    the foreign DNA in EE-GM1-   SEQ ID No. 14: nucleotide sequence comprising the 5′ region flanking    the foreign DNA in EE-GM2-   SEQ ID No. 15: nucleotide sequence comprising the 3′ region flanking    the foreign DNA in EE-GM2-   SEQ ID No. 16: primer SOY06-   SEQ ID No. 17: primer SOY07-   SEQ ID No. 18: primer SOY09-   SEQ ID No. 19: primer SOY010-   SEQ ID No. 20: nucleotide sequence of a foreign DNA and plant    flanking sequences in EE-GM3-   SEQ ID No. 21: primer SHA130-   SEQ ID No. 22: primer SHA178

EXAMPLES

1. Transformation of Glycine max with Herbicide Tolerance Genes.

1.1. Description of the Foreign DNA Comprising the 2mEPSPS andHPPD-Pf-W336 Chimeric Genes

Plasmid pSF10 is a pUC19 derived cloning vector which contains achimeric 2mepsps gene and a chimeric hppd-Pf-W336 gene located on a SalIfragment of about 7.3 kb. A full description of the DNA comprisedbetween the two SalI restriction sites is given in Table 1 below. Thenucleotide sequence is represented in SEQ ID No. 1.

TABLE 1 Nucleotide positions of the DNA comprised between the SalIrestriction sites of pSF10 (SEQ ID No 1) Nucleotide positionsOrientation Description and references  188-479 complement 3′nos:sequence including the 3′ untranslated region of the nopaline synthasegene from the T-DNA of pTiT37 of Agrobacterium tumefaciens (Depicker etal., 1982, Journal of Molecular and Applied Genetics, 1, 561-573) 480-1556 complement hppdPf W336: the coding sequence of the 4-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens strain A32modified by the replacement of the amino acid Glycine 336 with aTryptophane, as described by Boudec et al. (2001) U.S. Pat. No.6,245,968B1 1557-1928 complement TPotp Y: coding sequence of anoptimized transit peptide derivative (position 55 changed intoTyrosine), containing sequence of the RuBisCO small subunit genes of Zeamays (corn) and Helianthus annuus (sunflower), as described by Lebrun etal. (1996) U.S. Pat. No. 5,510,471 1929-2069 complement 5′tev: sequenceincluding the leader sequence of the tobacco etch virus as described byCarrington and Freed (1990) Journal of Virology, 64, 1590-1597 2070-3359complement Ph4a748 ABBC: sequence including the promoter region of thehistone H4 gene of Arabidopsis thaliana, containing an internalduplication (Chabouté et al., 1987) Plant Molecular Biology, 8, 179-191.3360-4374 Ph4a748: sequence including the promoter region of the histoneH4 gene of Arabidopsis thaliana (Chabouté et al., 1987) Plant MolecularBiology, 8, 179-191. 4375-4855 intron1 h3At: first intron of gene II ofthe histone H3.III variant of Arabidopsis thaliana (Chaubet et al.,1992) Journal of Molecular Biology, 225, 569-574. 4856-5227 TPotp C:coding sequence of the optimized transit peptide, containing sequence ofthe RuBisCO small subunit genes of Zea mays (corn) and Helianthus annuus(sunflower), as described by Lebrun et al. (1996) U.S. Pat. No.5,510,471 5228-6565 2mepsps: the coding sequence of the double- mutant5-enol-pyruvylshikimate-3-phosphate synthase gene of Zea mays (corn)(Lebrun et al., 1997) WO9704103-A 1 6566-7252 3′histonAt: sequenceincluding the 3′ untranslated region of the histone H4 gene ofArabidopsis thaliana (Chabouté et al., 1987) Plant Molecular Biology, 8,179-191.1.2. Event EE-GM3

The HPLC purified SalI-linearized pSF10 fragment of about 7.3 kb(containing the 2mEPSPS glyphosate-tolerance gene and the HPPD inhibitortolerance gene HPPD-Pf-W336) was used to obtain transformed soybeanplants by means of direct gene transfer into cells of soybean type Jack(Nickell, C. D., G. R. Noel, D. J. Thomas, and R. Waller. Registrationof ‘Jack’ soybean. Crop Sci 1365. 30.1990), followed by regeneration oftransformed plant cells into transgenic fertile soybean plants.

1.2.1 Identification of Elite Event EE-GM3

Elite event EE-GM3 was selected based on an extensive selectionprocedure based on good expression and stability of the herbicidetolerance genes, and its compatibility with optimal agronomiccharacteristics such as plant height, height to node, stand, vigor, seedyield, were evaluated. Soybean plants containing this event wereselected from a wide range of different transformation events obtainedusing the same chimeric genes. Parameters used in the selection of thisevent were: a) acceptable tolerance to isoxaflutole herbicideapplication in field trials, b) acceptable tolerance to glyphosateherbicide application in field trials, c) acceptable tolerance tocombined application of isoxaflutole and glyphosate herbicides in fieldtrials, d) an insertion of the herbicide tolerance transgenes at asingle locus in the soybean plant genome, with absence of vectorbackbone, e) overall agronomy similar to the parent plants used fortransformation (maturity, lodging, disease susceptibility, etc.), and f)no significant yield drag caused by the insertion of the transformingDNA (as compared to an isogenic line without the event, such as theplant line used for transformation, grown under the same conditions).

At the T3 generation, a homozygous line of the soybean transformationevent EE-GM3 was selected for seed production. Multi-location replicatedagronomic field studies were conducted in the regions of adaptation ofthe parent variety, Jack. Field evaluations included herbicide toleranceand agronomic performance. The agronomic performance of plantscontaining EE-GM3 was found comparable to Jack (when no herbicides wereapplied).

The field evaluations also showed that plants carrying the EE-GM3 eventhave:

-   -   similar plant morphology and seed characteristics compared to        Jack,    -   no change in response to soybean diseases compared to Jack, and    -   no changes in seed germination or dormancy compared to Jack.

Seed (T1 or S1 generation) harvested from the initial transformant (T0)plant (the plant transformed with the construct so as to produce eventEE-GM3) in the greenhouse were planted in the field. Three blocks wereplanted and sprayed with 0, 2, or 4 kg/ha glyphosate. Seed was harvestedfrom plants demonstrating the desired level of tolerance to theherbicide, glyphosate.

Seeds (T2 generation) harvested from self-pollinated T1 plants grown inthe field were sown “plant to row”. Chi square analysis of segregationdata for rows (fully or partially tolerant) and of individual plantswithin rows (tolerant or sensitive) demonstrates the expected Mendelianinheritance of a single insertion for EE-GM3.

Selection and seed increase continued until a line was determined to behomozygous for transformation event EE-GM3 and selected for core seedproduction in the fourth generation. Then, T5 generation seed served asa candidate for the development of different varieties. Plants in thesixth generation (T6 generation) were crossed with conventional soybeanbreeding lines in an introgression program designed to move the eventinto a broader base of commercial soybean germplasm. F1 hybrid plants(EE-GM3 lines×conventional lines) were grown to maturity and the F2 seedwas planted. Leaf samples of 901 F2 plants were analyzed by PCR primersdesigned to identify the zygosity of the EE-GM3 insert. The expectedratio of 1:2:1 for a single insertion segregation by the rules of Mendelwas observed.

The selected event EE-GM3 was introduced in different commercial geneticbackgrounds, and results of field trials on different locations werecompared. Plants were challenged with glyphosate herbicide and/orisoxaflutole herbicide using different treatments. The plants exhibitedgood herbicide tolerance. Hundreds of different soybean cultivarscontaining event EE-GM3 were used in an inheritance study, andherbicides were applied. Selected lines from this trial were laterincreased in the field and also treated with herbicide. From that trial,50 selected lines were increased, and these were also herbicide treated.The phytotoxicity scores for the latter lines sprayed with isoxaflutoleand glyphosate showed some variability in response, but the range ofresponses among the lines reflected similar variability as was observedacross 4 replications of the EE-GM3 event in the original Jackbackground, grown under the same treatment and environmental conditions.Hence, tolerance to the relevant herbicides across a broad range ofgermplasm was observed for plants comprising EE-GM3.

Furthermore, plants containing the event EE-GM3 had normal leaf, flowerand pod morphology, excellent fertility, and showed no disease orabnormal insect susceptibility in multiple genetic backgrounds. Duringintrogression into multiple genetic backgrounds no aberrant problems orabnormalities were observed.

In one season, a 10 location study was designed to compare the agronomicperformance of double herbicide tolerant soybean comprisingtransformation event EE-GM3 to the transformation parent variety, Jackand some non-transgenic soybean varieties. Using a randomized completeblock design, EE-GM3 plants were grown in replicated plots with eitherconventional weed control or with the intended herbicides, glyphosateand isoxaflutole. Plots with soybean plants containing transformationevent EE-GM3 were sprayed with isoxaflutole herbicide at a target rateof 70 grams ai/Ha and with glyphosate herbicide at a target rate of 1060grams ai/Ha. Herbicide application was made to these plants as a foliarspray at about the V4-V5 plant growth stage. Agronomic observations weremade in the early, mid and late season. The plant density (parameter;stand count) was higher for the Jack and the non-transgenic varietyplots than in the event EE-GM3 plots by one standard deviation. Theearly stand count difference may have been the result of seed lotquality, as the EE-GM3 planting seed was produced in counter seasonnursery, while the seed of the non-transgenic varieties was produced inthe contiguous US, normal production season. However, the number of daysto achieve 50% emergence and the plant vigor ratings were the same,indicating that the seed lots were comparable for these performanceparameters. In the late season stand counts, Jack and the non-transgenicvarieties remained different by one standard deviation from EE-GM3plants. Plot yields of EE-GM3 event plants were also lower than those ofJack by one standard deviation, perhaps a result of the lower plantdensity of the EE-GM3 event plots. The yield of the non-transgenicvarieties was more than Jack as could be expected because of theadvancement in yield potential found in more recent varieties.

In one trial, plant health ratings were made at three growth stages:V4-5, R1 and full maturity. The first evaluation was shortly after theintended herbicide application. At the time of the final plant healthevaluation, the EE-GM3-containing plants sprayed with both herbicideshad the same score as the unsprayed Jack plants, or the unsprayed plantscomprising EE-GM3. In ratings by the agronomic staff, theherbicide-sprayed plants received a health rating of 3-4 (moderateinjury) at the V4-5 and R1 plant growth stages. The unsprayed plants(untransformed Jack or soybean plants containing EE-GM3) were rated as4.6-4.8 (rating of 5 indicates no injury). At the final plant healthrating, all the plots received the same rating of 5 (no injury).

One trial season was one of exceptional rainfall and crop injury in theEE-GM3 plants following the intended herbicide was more obvious thanobserved in other seasons. The field evaluations also includedmonitoring of the fitness characters (reproduction, disease resistance,fecundity, seed dispersal, dormancy, persistence). For the reproductivecharacteristics; days to emergence, days to 50% flowering and days to90% pods maturing, the EE-GM3 and Jack plants were not different. Nodifference was noted in the reaction to natural infestations of plantdiseases and insect pests. Although EE-GM3 produced less ultimate yieldthan Jack, no difference in fecundity (100 seed weight) was found. Theassessment of seed dispersal parameters (pod shattering and plantlodging) found EE-GM3 and Jack to have the same pod shattering score,but found EE-GM3 plants to be less prone to lodging. Evaluation of seedharvested from the 10 locations found no concerns raised by germinationor dormancy testing.

In these trials during the season with exceptional rainfall, the finalyield of EE-GM3 plants, regardless of the weed control treatment, wasless than the yield of Jack by one standard deviation (perhaps a resultof the lower plant density of the EE-GM3 event plots). In theexceptionally wet season, crop injury (bleaching in 10-30% of the croparea) was reported for EE-GM3 plots up to six weeks following foliarapplication of the glyphosate and isoxaflutole herbicides. However, bymaturity, “no injury” plant health ratings were assigned to all theplots. Replicated multi-location field trials with EE-GM3 introgressedin elite soybean cultivar background, when compared to near-isogenicsister lines not containing the transgene, are expected to show no yielddifference between plants containing event EE-GM3 and the near-isogeniclines (in the absence of herbicide treatment).

Further, in a replicated field trial significant crop tolerance(bleaching of less than 10%) was found in soybean plants comprisingEE-GM3 when treated either pre- or post-emergence with IFT (70 gr ai/hawith 0.5% NIS, Agridex), but also significant crop tolerance (bleachingof less than 10%) was found in soybean plants comprising EE-GM3 whentreated with a post-emergence application of pyrasulfotole (35 gr ai/hawith 0.5% NIS, Agridex), another HPPD inhibitor herbicide.

1.2.2 Identification of the Flanking Regions and Foreign DNA of EliteEvent EE-GM3

The sequence of the regions flanking the foreign DNA comprising theherbicide tolerance genes in the EE-GM3 elite event was determined to beas follows:

1.2.2.1. Right (5′) Flanking Region

The fragment identified as comprising the 5′ flanking region wassequenced and its nucleotide sequence is represented in SEQ ID No. 2.The sequence between nucleotide 1 and 1451 corresponds to plant DNA,while the sequence between nucleotide 1452 and 1843 corresponds toforeign DNA.

1.2.2.2. Left (3′) Flanking Region

The fragment identified as comprising the 3′ flanking region wassequenced and its nucleotide sequence is represented in SEQ ID No. 3.The sequence between nucleotide 1 and 240 corresponds to foreign DNA,while the sequence between nucleotide 241 and 1408 corresponds to plantDNA.

1.2.2.3. Foreign DNA Comprising the Herbicide Tolerance Genes of EE-GM3

Using different molecular techniques, it has been determined that theforeign DNA of elite event EE-GM3 comprising the herbicide tolerancegenes contains two partial 3′ histonAt sequences in a head-to-headorientation, followed by 2 almost complete copies of the SalI fragmentof pSF10 arranged in head-to-tail orientation (see FIG. 1).

The foreign DNA comprising the herbicide tolerance genes of EE-GM3 thuscontains in order the following sequences:

-   -   from nucleotide 1 to nucleotide 199: the nucleotide sequence        corresponding to complement of the nucleotide sequence of SEQ ID        1 from nt 6760 to nt 6958;    -   from nucleotide 200 to nucleotide 624: the nucleotide sequence        corresponding to the nucleotide sequence of SEQ ID 1 from nt        6874 to nt 7298;    -   from nucleotide 625 to nucleotide 7909: the nucleotide sequence        corresponding to the nucleotide sequence of SEQ ID 1 from nt 7        to nt 7291;    -   from nucleotide 7910 to nucleotide 15163: the nucleotide        sequence corresponding to the nucleotide sequence of SEQ ID 1        from nt 12 to nt 7265; and    -   from nucleotide 15164 to nucleotide 15187: the nucleotide        sequence corresponding to the nucleotide sequence of SEQ ID 3        from nt 217 to nt 240 (this sequence does not correspond to        either pSF10 plasmid DNA or wt plant DNA and therefore is        designated filler DNA).

This foreign DNA is preceded immediately upstream and contiguous withthe foreign DNA by the 5′ flanking sequence of SEQ ID No 2 fromnucleotide 1 to 1451 and is followed immediately downstream andcontiguous with the foreign DNA by the 3′ flanking sequence of SEQ ID No3 from nucleotide 241 to nucleotide 1408.

Confirmed full DNA sequencing of the foreign DNA and flanking DNAsequences in EE-GM3 resulted in the sequence reported in SEQ ID No. 20.In this sequence, the inserted DNA is from nucleotide position 1452 tonucleotide position 16638, and the 2 almost complete copies from pSF10arranged in head-to-tail orientation are from nucleotide position 2257to nucleotide position 16601. The 5′ flanking DNA sequence in SEQ ID No.20 is the sequence from nucleotide position 1 to nucleotide position1451 in SEQ ID No. 20, and the 3′ flanking DNA sequence in SEQ ID No. 20is the sequence from nucleotide position 16639 to nucleotide position17806 in SEQ ID No. 20.

1.3. Description of the Foreign DNA Comprising thePhosphinothricinacetyltransferase Chimeric Genes

Plasmid pB2/P35SAcK is a pUC19 derived cloning vector which contains achimeric pat gene. A description of the vector is given in Table 2below. The nucleotide sequence thereof is represented in SEQ ID No. 11.

TABLE 2 Nucleotide positions of constituents of pB2/P35SAcK (SEQ ID No11) Nucleotide positions Description and references  461-1003 35Spromotor from Cauliflower Mosaic Virus 1004-1011 Synthetic polylinkerderived sequences 1012-1563 Synthetic pat gene (amino acid sequence fromStreptomyces viridochromogenes) 1564-1581 Synthetic polylinker derivedsequences 1582-1784 35S terminator from Cauliflower Mosaic Virus1.4. Event EE-GM11.4.1 Identification of EE-GM1

Herbicide-resistant soybean was developed by transformation of soybeanwith vector pB2/P35SAcK using direct gene transfer.

Elite event EE-GM1 was selected based on an extensive selectionprocedure based on good expression and stability of the herbicideresistance gene and its compatibility with optimal agronomiccharacteristics.

1.4.2 Identification of the Flanking Regions and Foreign DNA of EliteEvent EE-GM1

1.4.2.1. Right (5′) Flanking Region

The fragment identified as comprising the 5′ flanking region wassequenced and its nucleotide sequence is represented in SEQ ID No. 12.The sequence between nucleotide 1 and 209 corresponds to plant DNA,while the sequence between nucleotide 210 and 720 corresponds to foreignDNA.

1.4.2.2. Left (3′) Flanking Region

The fragment identified as comprising the 3′ flanking region wassequenced and its nucleotide sequence is represented in SEQ ID No. 13.The sequence between nucleotide 1 and 568 corresponds to foreign DNA,while the sequence between nucleotide 569 and 1000 corresponds to plantDNA.

1.4.2.3. Foreign DNA Comprising the Herbicide Tolerance Genes of EE-GM1

Using different molecular techniques, it has been determined that theforeign DNA of elite event EE-GM1 comprising the herbicide tolerancegene comprises two copies of the chimeric pat gene in a direct repeatstructure (see FIG. 3).

The foreign DNA comprising the herbicide tolerance genes of EE-GM1 thuscontains in order the following sequences:

-   -   from nucleotide 1 to nucleotide 3122: the nucleotide sequence        corresponding to the nucleotide sequence of SEQ ID 11 from nt        340 to nt 3461;    -   from nucleotide 3123 to nucleotide 3458: the nucleotide sequence        corresponding to the complement of the nucleotide sequence of        SEQ ID 11 from nt 1 to nt 336;    -   from nucleotide 3459 to nucleotide 4073: the nucleotide sequence        corresponding to the complement of the nucleotide sequence of        SEQ ID 11 from nt 3462 to nt 4076; and    -   from nucleotide 4074 to nucleotide 6780: the nucleotide sequence        corresponding to the nucleotide sequence of SEQ ID 11 from nt        337 to nt 3043; and from nucleotide 6781 to nucleotide 6790: the        nucleotide sequence corresponding to the nucleotide sequence of        SEQ ID 13 from nt 559 to nt 568 (this sequence does not        correspond to either plasmid DNA or wt plant DNA and therefore        is designated filler DNA).

This foreign DNA of EE-GM1 is preceded immediately upstream andcontiguous with the foreign DNA at the 5′ flanking sequence of SEQ ID No12 from nucleotide 1 to 209 and is followed immediately downstream andcontiguous with the foreign DNA at the 3′ flanking sequence of SEQ ID No13 from nucleotide 569 to nucleotide 1000.

1.5. Event EE-GM2

Herbicide-resistant soybean was developed by transformation of soybeanwith vector pB2/P35SAcK using direct gene transfer.

Elite event EE-GM2 was selected based on an extensive selectionprocedure based on good expression and stability of the herbicideresistance gene and its compatibility with optimal agronomiccharacteristics.

1.5.1. Identification of the Flanking Regions and Foreign DNA of EliteEvent EE-GM2

1.5.1.1. Right (5′) Flanking Region

The fragment identified as comprising the 5′ flanking region wassequenced and its nucleotide sequence is represented in SEQ ID No. 14.The sequence between nucleotide 1 and 311 corresponds to plant DNA,while the sequence between nucleotide 312 and 810 corresponds to foreignDNA.

1.5.1.2. Left (3′) Flanking Region

The fragment identified as comprising the 3′ flanking region wassequenced and its nucleotide sequence is represented in SEQ ID No. 15.The sequence between nucleotide 1 and 507 corresponds to foreign DNA,while the sequence between nucleotide 569 and 1880 corresponds to plantDNA.

1.5.1.3. Foreign DNA Comprising the Herbicide Tolerance Genes of EE-GM2

Using different molecular techniques, it has been determined that theforeign DNA of elite event EE-GM2 comprising the herbicide tolerancegene comprises one copy of the chimeric pat gene (see FIG. 4).

The foreign DNA comprising the herbicide tolerance genes of EE-GM2 thuscontains in order the following sequences:

-   -   from nucleotide 1 to nucleotide 391: the nucleotide sequence        corresponding to the nucleotide sequence of SEQ ID 11 from nt        3458 to nt 3848; and    -   from nucleotide 392 to nucleotide 3436: the nucleotide sequence        corresponding to the nucleotide sequence of SEQ ID 11 from nt        413 to nt 3457.

This foreign DNA of EE-GM2 is preceded immediately upstream andcontiguous with the foreign DNA at the 5′ flanking sequence of SEQ ID No14 from nucleotide 1 to 311 and is followed immediately downstream andcontiguous with the foreign DNA at the 3′ flanking sequence of SEQ ID No15 from nucleotide 508 to nucleotide 1880.

2. Development of Polymerase Chain Reaction Identification Protocols forEE-GM3.

2.1. Primers

Specific primers were developed which recognize sequences within theelite event.

A primer was developed which recognizes a sequence within the 3′flanking region of EE-GM3. A second primer was then selected within thesequence of the foreign DNA so that the primers span a sequence of about263 nucleotides. The following primers were found to give particularlyclear and reproducible results in a PCR reaction on EE-GM3 DNA:

(SEQ ID No.: 4) SOY028: 5′-ATC.gCT.TTA.ACg.TCC.CTC.Ag -3(target: insert DNA) (SEQ ID No.: 5) SOY029:5′-CAA.ggC.CTC.gAg.ATT.ATC -3′ (target: plant DNA)

Primers targeting an endogenous sequence are preferably included in thePCR cocktail. These primers serve as an internal control in unknownsamples and in the DNA positive control. A positive result with theendogenous primer-pair (presence of an PCR amplified fragment of 413 bp)demonstrates that there is ample DNA of adequate quality in the genomicDNA preparation for a PCR product to be generated. The endogenousprimers were selected to recognize the endogenous actin soybean gene:

(SEQ ID No.: 9) SOY01 5′-gTC.AgC.CAC.ACA.gTg.CCT.AT -3′ (SEQ ID No.: 10)SOY02 5′-gTT.ACC.gTA.CAg.gTC.TTT.CC -3′2.2. Amplified Fragments

The expected amplified fragments in the PCR reaction are:

-   For primer pair SOY01-SOY02: 413 bp (endogenous control)-   For primer pair SOY028-SOY029: 263 bp (EE-GM3 elite event)    2.3. Template DNA

Template DNA was prepared from a leaf punch according to Edwards et al.(Nucleic Acid Research, 19, p 1349, 1991). When using DNA prepared withother methods, a test run utilizing different amounts of template shouldbe done. Usually 50 ng of genomic template DNA yields the best results.

2.4. Assigned Positive and Negative Controls

To avoid false positives or negatives, it was determined that thefollowing positive and negative controls should be included in a PCRrun:

-   -   Master Mix control (DNA negative control). This is a PCR in        which no DNA is added to the reaction. When the expected result,        no PCR products, is observed this indicates that the PCR        cocktail was not contaminated with target DNA.    -   A DNA positive control (genomic DNA sample known to contain the        transgenic sequences). Successful amplification of this positive        control demonstrates that the PCR was run under conditions which        allow for the amplification of target sequences.    -   A wild-type DNA control. This is a PCR in which the template DNA        provided is genomic DNA prepared from a non-transgenic plant.        When the expected result, no amplification of a transgene PCR        product but amplification of the endogenous PCR product, is        observed this indicates that there is no detectable transgene        background amplification in a genomic DNA sample.        2.5. PCR Conditions

Optimal results were obtained under the following conditions (Indescribing the various conditions for optimal results is meant toprovide examples of such conditions. Clearly one skilled in the artcould vary conditions, reagents and parameters such as using other Taqpolymerases, and achieve desirable results):

-   -   the PCR mix for 25 μl reactions contains:        -   20 ng template DNA        -   2.5 μl 10× Amplification Buffer (supplied by the            manufacturer with the Taq polymerase)        -   0.5 μl 10 mM dNTP's        -   0.4 μl SOY01 (10 pmoles/μl)        -   0.4 μA SOY02 (10 pmoles/μl)        -   0.7 μA SOY028 (10 pmoles/μl)        -   0.7 μA SOY029 (10 pmoles/μl)        -   0.1 μA Taq DNA polymerase (5 units/μl)        -   water up to 25 μl    -   the thermocycling profile to be followed for optimal results is        the following:        -   4 min. at 95° C.    -    Followed by: 1 min. at 95° C.        -   1 min. at 57° C.        -   2 min. at 72° C.        -   For 5 cycles    -    Followed by: 30 sec. at 92° C.        -   30 sec. at 57° C.        -   1 min. at 72° C.        -   For 25 cycles    -    Followed by: 10 minutes at 72° C.        2.6. Agarose Gel Analysis

To optimally visualize the results of the PCR it was determined thatbetween 10 and 20 μl of the PCR samples should be applied on a 1.5%agarose gel (Tris-borate buffer) with an appropriate molecular weightmarker (e.g. 100 bp ladder Pharmacia).

2.7. Validation of the Results

It was determined that data from transgenic plant DNA samples within asingle PCR run and a single PCR cocktail should not be acceptableunless 1) the DNA positive control shows the expected PCR products(transgenic and endogenous fragments), 2) the DNA negative control isnegative for PCR amplification (no fragments) and 3) the wild-type DNAcontrol shows the expected result (endogenous fragment amplification).

When following the PCR Identification Protocol for EE-GM3 as describedabove, lanes showing visible amounts of the transgenic and endogenousPCR products of the expected sizes, indicate that the correspondingplant from which the genomic template DNA was prepared, has inheritedthe EE-GM3 elite event. Lanes not showing visible amounts of either ofthe transgenic PCR products and showing visible amounts of theendogenous PCR product, indicate that the corresponding plant from whichthe genomic template DNA was prepared, does not comprise the eliteevent. Lanes not showing visible amounts of the endogenous andtransgenic PCR products, indicate that the quality and/or quantity ofthe genomic DNA didn't allow for a PCR product to be generated. Theseplants cannot be scored. The genomic DNA preparation should be repeatedand a new PCR run, with the appropriate controls, has to be performed.

2.8. Use of Discriminating PCR Protocol to Identify EE-GM3

Before attempting to screen unknowns, a test run, with all appropriatecontrols, is performed. The developed protocol might requireoptimization for components that may differ between labs (template DNApreparation, Taq DNA polymerase, quality of the primers, dNTP's,thermocyler, etc.).

Amplification of the endogenous sequence plays a key role in theprotocol. One has to attain PCR and thermocycling conditions thatamplify equimolar quantities of both the endogenous and the transgenicsequence in a known transgenic genomic DNA template. Whenever thetargeted endogenous fragment is not amplified or whenever the targetedsequences are not amplified with the same ethidium bromide stainingintensities, as judged by agarose gel electrophoresis, optimization ofthe PCR conditions may be required.

Leaf material from a number of soybean plants, some of which comprisingEE-GM3 were tested according to the above-described protocol. Samplesfrom elite event EE-GM3 and from soybean wild-type were taken aspositive and negative controls, respectively.

FIG. 2 illustrates the result obtained with the elite event PCRIdentification Protocol for EE-GM3 on a number of soybean plant samples.The samples in lanes 2 and 3 were found to contain elite event EE-GM3 asthe 263 bp band is detected, while the samples in lanes 4 to 8 do notcomprise EE-GM3. Lanes 6 and 7 comprise samples from other soybeantransformation events obtained using the same herbicide tolerancechimeric genes; lane 8 contains DNA from wild type soybean plants andlane 9 represents the negative control (water) sample, lanes 1 and 10represent the Molecular Weight Marker (100 bp ladder).

2.9. dPCR Assay for EE-GM3 Detection in Bulked Seed

A discriminating PCR (dPCR) assay is set up to detect low level presenceof EE-GM3 in bulked seeds. A minimum level of 0.4% (w/w) of transgenicseeds in a bulk of non transgenic seeds was successfully detected underrepeatable conditions. Therefore the Limit of Detection is determined tobe 0.4% (w/w).

The following primers are applied in this target PCR reaction:

-   Forward Primer Targeted to the T-DNA Sequence:

(SED ID No. 12) SHA130 5′ -CTA.TAT.TCT.ggT.TCC.AAT.TTA.TC -3′

-   Reverse Primer Targeted to the 3′ Flanking Sequence:

(SEQ ID No. 13) SMP178 5′ -TgA.ggC.ACg.TAT.TgA.TgA.CC -3′

The expected amplified fragment in the PCR reaction from these primersis 115 bp.

The target PCR reaction is performed on approximately 200 ng of templateDNA prepared from ground bulked seed according to a modified GentraPuregene DNA purification extraction kit (Qiagen). When using DNAprepared with other methods, a test run using samples with knownrelative levels of EE-GM3 should be performed.

A validated reference system PCR reaction, targeting an endogenoussequence, should ideally be performed in a separate PCR run to verifythe suitability of the DNA sample for PCR analysis to avoid falsenegative results.

For unknown test samples the PCR experiment should ideally include theappropriate positive and negative control samples, i.e.:

-   -   Master Mix control (DNA negative control). This is a PCR in        which no DNA is added to the reaction. When the expected result        (no PCR product) is observed for both the target and the        reference system reaction this indicates that the PCR cocktail        was not contaminated with target DNA.    -   A DNA positive control (genomic DNA sample known to contain the        transgenic sequences). Successful amplification of this positive        control demonstrates that the PCR was run under conditions which        allow for the amplification of target sequences.    -   Also a wild-type DNA control can be added in this PCR. This is a        PCR in which the template

DNA provided is genomic DNA prepared from a non-transgenic plant. Whenthe expected result, no amplification of a transgene PCR product butamplification of the endogenous PCR product, is observed this indicatesthat there is no detectable transgene background amplification in agenomic DNA sample.

Optimal results are obtained under the following conditions:

-   -   the PCR mix for 25 μl reactions contains:        -   200 ng template DNA        -   5 μl 5× Reaction Buffer        -   0.25 μl 20 mM dNTP's        -   0.7 μl SHA130 (10 pmoles/l)        -   0.4 μl SMP178 (10 pmoles/l)        -   0.1 μl GO-Taq DNA polymerase (5 units/l)        -   Add water up to 25 μl    -   the thermocycling profile to be followed for optimal results is        the following:        -   4 min. at 95° C.    -    Followed by: 1 min. at 95° C.        -   1 min. at 57° C.        -   2 min. at 72° C.        -   For 5 cycles    -    Followed by: 30 sec. at 92° C.        -   30 sec. at 57° C.        -   1 min. at 72° C.        -   For 30 cycles    -    Followed by: 10 minutes at 72° C.

To optimally visualize the results of the PCR it was determined that 25μl of the PCR product should be applied on a 1.5% agarose gel(Tris-borate buffer) with an appropriate molecular weight marker (e.g.50 bp ladder).

When following the PCR method as described above, lanes showing visibleamounts of the target and reference system PCR products of the expectedsizes, indicate that the test sample from which the genomic template DNAwas prepared, contained levels of EE-GM3 elite event above the detectionlimit of the target reaction

Lanes not showing visible amounts of the target PCR products but showingvisible amounts of the reference system PCR product, indicate that thetest sample from which the genomic template DNA was prepared, containedlevels of EE-GM3 elite event below the detection limit of the targetreaction

Lanes not showing visible amounts of the endogenous and transgenic PCRproducts, indicate that the quality and/or quantity of the genomic DNAdidn't allow for a PCR product to be generated. These plants cannot bescored. The genomic DNA preparation should be repeated and a new PCRrun, with the appropriate controls, has to be performed.

3. Use of a Specific Integration Fragment as a Probe for Detection ofMaterial Comprising EE-GM3.

A specific integration fragment of EE-GM3 is obtained by PCRamplification using specific primers SOY028 (SEQ ID No. 4) and SOY029(SEQ ID No. 5) yielding an amplicon with the nucleotide sequence of SEQID No 8 or by chemical synthesis and is labeled. This integrationfragment is used as a specific probe for the detection of EE-GM3 inbiological samples. Nucleic acid is extracted from the samples accordingto standard procedures. This nucleic acid is then contacted with thespecific probe under hybridization conditions which are optimized toallow formation of a hybrid. The formation of the hybrid is thendetected to indicate the presence of EE-GM3 nucleic acid in the sample.Optionally, the nucleic acid in the samples is amplified using thespecific primers prior to contact with the specific probe.Alternatively, the nucleic acid is labeled prior to contact with thespecific probe instead of the integration fragment. Optionally, thespecific probe is attached to a solid carrier (such as, but not limitedto a filter, strip or beads), prior to contact with the samples.

4. Polymerase Chain Reaction Identification Protocol for EE-GM1.

4.1. Primers

Specific primers were developed which recognize sequences within theelite event. More particularly, a primer was developed which recognizesa sequence within the 5′ flanking region of EE-GM1. A second primer wasthen selected within the sequence of the foreign DNA so that the primersspan a sequence of about 183 nucleotides. The following primers werefound to give particularly clear and reproducible results in a PCRreaction on EE-GM1 DNA:

(SEQ ID No.: 16) SOY06: 5′- ggC.gTT.CgT.AgT.gAC.TgA.gg -3′(target: plant DNA) (SEQ ID No.: 17) SOY07:5′-gTT.TTA.CAA.CgT.CgT.gAC.Tgg-3′ (target: insert DNA)

Primers targeting an endogenous sequence are preferably included in thePCR cocktail. These primers serve as an internal control in unknownsamples and in the DNA positive control. A positive result with theendogenous primer-pair demonstrates that there is ample DNA of adequatequality in the genomic DNA preparation for a PCR product to begenerated. The endogenous primers were selected to recognize ahousekeeping gene in Glycine max:

(SEQ ID No.: 9) SOY01: 5′-gTC.AgC.CAC.ACA.gTg.CCT.AT-3′(located in Glycine max actin 1 gene (Accession J01298))(SEQ ID No.: 10) SOY02: 5′-gTT.ACC.gTA.CAg.gTC.TTT.CC-3′(located in Glycine max actin 1 gene (Accession J01298))4.2. Amplified Fragments

The expected amplified fragments in the PCR reaction are:

-   For primer pair SOY01-SOY02: 413 bp (endogenous control)-   For primer pair SOY06-SOY07: 183 bp (EE-GM1 elite Event)    4.3. Template DNA

Template DNA was prepared from a leaf punch according to Edwards et al.(Nucleic Acid Research, 19, p 1349, 1991). When using DNA prepared withother methods, a test run utilizing different amounts of template shouldbe done. Usually 50 ng of genomic template DNA yields the best results.

4.4. Assigned Positive and Negative Controls

To avoid false positives or negatives, it was determined that thefollowing positive and negative controls should be included in a PCRrun:

-   -   Master Mix control (DNA negative control). This is a PCR in        which no DNA is added to the reaction. When the expected result,        no PCR products, is observed this indicates that the PCR        cocktail was not contaminated with target DNA.    -   A DNA positive control (genomic DNA sample known to contain the        transgenic sequences). Successful amplification of this positive        control demonstrates that the PCR was run under conditions which        allow for the amplification of target sequences.    -   A wild-type DNA control. This is a PCR in which the template DNA        provided is genomic DNA prepared from a non-transgenic plant.        When the expected result, no amplification of a transgene PCR        product but amplification of the endogenous PCR product, is        observed this indicates that there is no detectable transgene        background amplification in a genomic DNA sample.        4.5. PCR Conditions

Optimal results were obtained under the following conditions:

-   -   the PCR mix for 25 μl reactions contains:        -   2.5 μl template DNA        -   2.5 μl 10× Amplification Buffer (supplied with Taq            polymerase)        -   0.5 μl 10 mM dNTP's        -   0.5 μl SOY06 (10 pmoles/μl)        -   0.5 μl SOY07 (10 pmoles/μl)        -   0.25 μl SOY01 (10 pmoles/μl)        -   0.25 μl SOY02 (10 pmoles/μl)        -   0.1 μl Taq DNA polymerase (5 units/μl)    -   water up to 25 μl    -   the thermocycling profile to be followed for optimal results is        the following:        -   4 min. at 95° C.    -    Followed by: 1 min. at 95° C.        -   1 min. at 57° C.        -   2 min. at 72° C.        -   For 5 cycles    -    Followed by: 30 sec. at 92° C.        -   30 sec. at 57° C.        -   1 min. at 72° C.        -   For 25 cycles    -    Followed by: 5 minutes at 72° C.        4.6. Agarose Gel Analysis

To optimally visualize the results of the PCR it was determined thatbetween 10 and 20 μl of the PCR samples should be applied on a 1.5%agarose gel (Tris-borate buffer) with an appropriate molecular weightmarker (e.g. 100 bp ladder Pharmacia).

4.7. Validation of the Results

It was determined that data from transgenic plant DNA samples within asingle PCR run and a single PCR cocktail should not be acceptableunless 1) the DNA positive control shows the expected PCR products(transgenic and endogenous fragments), 2) the DNA negative control isnegative for PCR amplification (no fragments) and 3) the wild-type DNAcontrol shows the expected result (endogenous fragment amplification).

When following the PCR Identification Protocol for EE-GM1 as describedabove, lanes showing visible amounts of the transgenic and endogenousPCR products of the expected sizes, indicate that the correspondingplant from which the genomic template DNA was prepared, has inheritedthe EE-GM1 elite event. Lanes not showing visible amounts of either ofthe transgenic PCR products and showing visible amounts of theendogenous PCR product, indicate that the corresponding plant from whichthe genomic template DNA was prepared, does not comprise the eliteevent. Lanes not showing visible amounts of the endogenous andtransgenic PCR products, indicate that the quality and/or quantity ofthe genomic DNA didn't allow for a PCR product to be generated. Theseplants cannot be scored. The genomic DNA preparation should be repeatedand a new PCR run, with the appropriate controls, has to be performed.

4.8. Use of Discriminating PCR Protocol to Identify EE-GM1

Before attempting to screen unknowns, a test run, with all appropriatecontrols, has to be performed. The developed protocol might requireoptimization for components that may differ between labs (template DNApreparation, Taq DNA polymerase, quality of the primers, dNTP's,thermocyler, etc.).

Amplification of the endogenous sequence plays a key role in theprotocol. One has to attain PCR and thermocycling conditions thatamplify equimolar quantities of both the endogenous and transgenicsequence in a known transgenic genomic DNA template. Whenever thetargeted endogenous fragment is not amplified or whenever the targetedsequences are not amplified with the same ethidium bromide stainingintensities, as judged by agarose gel electrophoresis, optimization ofthe PCR conditions may be required.

Glycine max leaf material from a number of plants, some of whichcomprising EE-GM1 were tested according to the above-described protocol.Samples from elite event EE-GM1 and from Glycine max wild-type weretaken as positive and negative controls, respectively.

FIG. 5 illustrates the result obtained with the elite event PCRIdentification Protocol for EE-GM1 on a number of soybean plant samples(lanes 1 to 14). The samples in lane 1 were found to contain the eliteevent as the 185 bp band is detected, while the samples in lanes 2, 3and 4 do not comprise EE-GM1. Lane 2 comprises another soybean eliteevent, lane 3 represents a non-transgenic Glycine max control; lane 4represents the negative control (water) sample, and lane 5 representsthe Molecular Weight Marker (100 bp).

5. Use of a Specific Integration Fragment as a Probe for Detection ofMaterial Comprising EE-GM1.

A specific integration fragment of EE-GM1 is obtained by PCRamplification using specific primers SOY06 (SEQ ID No. 16) and SOY07(SEQ ID No. 17) or by chemical synthesis and is labeled. Thisintegration fragment is used as a specific probe for the detection ofEE-GM1 in biological samples. Nucleic acid is extracted from the samplesaccording to standard procedures. This nucleic acid is then contactedwith the specific probe under hybridization conditions which areoptimized to allow formation of a hybrid. The formation of the hybrid isthen detected to indicate the presence of EE-GM1 nucleic acid in thesample. Optionally, the nucleic acid in the samples is amplified usingthe specific primers prior to contact with the specific probe.Alternatively, the nucleic acid is labeled prior to contact with thespecific probe instead of the integration fragment. Optionally, thespecific probe is attached to a solid carrier (such as, but not limitedto a filter, strip or beads), prior to contact with the samples.

6. Polymerase Chain Reaction Identification Protocol for EE-GM2.

6.1. Primers

Specific primers were developed which recognize sequences within theelite event. More particularly, a primer was developed which recognizesa sequence within the 3′ flanking region of EE-GM2. A second primer wasthen selected within the sequence of the foreign DNA so that the primersspan a sequence of about 150 nucleotides. The following primers werefound to give particularly clear and reproducible results in a PCRreaction on EE-GM2 DNA:

(SEQ ID No.: 18) SOY09: 5′- TgT.ggT.TAT.ggC.ggT.gCC.ATC -3′(target: plant DNA) (SEQ ID No.: 19) SOY010:5′-TgC.TAC.Agg.CAT.CgT.ggT.gTC-3′ (target: insert DNA)

Primers targeting an endogenous sequence are preferably included in thePCR cocktail. These primers serve as an internal control in unknownsamples and in the DNA positive control. A positive result with theendogenous primer-pair demonstrates that there is ample DNA of adequatequality in the genomic DNA preparation for a PCR product to begenerated. The endogenous primers were selected to recognize ahousekeeping gene in Glycine max:

(SEQ ID No.: 9) SOY01: 5′-gTC.AgC.CAC.ACA.gTg.CCT.AT-3′(located in Glycine max actin 1 gene (Accession J01298))(SEQ ID No.: 10) SOY02: 5′-gTT.ACC.gTA.CAg.gTC.TTT.CC-3′(located in Glycine max actin 1 gene (Accession J01298))6.2. Amplified Fragments

The expected amplified fragments in the PCR reaction are:

-   For primer pair SOY01-SOY02: 413 bp (endogenous control)-   For primer pair SOY09-SOY010: 151 bp (EE-GM2 elite Event)    6.3. Template DNA

Template DNA was prepared from a leaf punch according to Edwards et al.(Nucleic Acid Research, 19, p 1349, 1991). When using DNA prepared withother methods, a test run utilizing different amounts of template shouldbe done. Usually 50 ng of genomic template DNA yields the best results.

6.4. Assigned Positive and Negative Controls

To avoid false positives or negatives, it was determined that thefollowing positive and negative controls should be included in a PCRrun:

-   -   Master Mix control (DNA negative control). This is a PCR in        which no DNA is added to the reaction. When the expected result,        no PCR products, is observed this indicates that the PCR        cocktail was not contaminated with target DNA.    -   A DNA positive control (genomic DNA sample known to contain the        transgenic sequences). Successful amplification of this positive        control demonstrates that the PCR was run under conditions which        allow for the amplification of target sequences.    -   A wild-type DNA control. This is a PCR in which the template DNA        provided is genomic DNA prepared from a non-transgenic plant.        When the expected result, no amplification of a transgene PCR        product but amplification of the endogenous PCR product, is        observed this indicates that there is no detectable transgene        background amplification in a genomic DNA sample.        6.5. PCR Conditions

Optimal results were obtained under the following conditions:

-   -   the PCR mix for 25 μl reactions contains:        -   2.5 μl template DNA        -   2.5 μl 10× Amplification Buffer (supplied with Taq            polymerase)        -   0.5 μl 10 mM dNTP's        -   0.5 μl SOY09 (10 pmoles/μl)        -   0.5 μl SOY010 (10 pmoles/μl)        -   0.25 μl SOY01 (10 pmoles/μl)        -   0.25 μl SOY02 (10 pmoles/μl)        -   0.1 μl Taq DNA polymerase (5 units/μl)        -   water up to 25 μl    -   the thermocycling profile to be followed for optimal results is        the following:        -   4 min. at 95° C.    -    Followed by: 1 min. at 95° C.        -   1 min. at 57° C.        -   2 min. at 72° C.        -   For 5 cycles    -    Followed by: 30 sec. at 92° C.        -   30 sec. at 57° C.    -    Followed by: 5 minutes at 72° C.        6.6. Agarose Gel Analysis

To optimally visualize the results of the PCR it was determined thatbetween 10 and 20 μl of the PCR samples should be applied on a 1.5%agarose gel (Tris-borate buffer) with an appropriate molecular weightmarker (e.g. 100 bp ladder Pharmacia).

6.7. Validation of the Results

It was determined that data from transgenic plant DNA samples within asingle PCR run and a single PCR cocktail should not be acceptableunless 1) the DNA positive control shows the expected PCR products(transgenic and endogenous fragments), 2) the DNA negative control isnegative for PCR amplification (no fragments) and 3) the wild-type DNAcontrol shows the expected result (endogenous fragment amplification).

When following the PCR Identification Protocol for EE-GM2 as describedabove, lanes showing visible amounts of the transgenic and endogenousPCR products of the expected sizes, indicate that the correspondingplant from which the genomic template DNA was prepared, has inheritedthe EE-GM2 elite event. Lanes not showing visible amounts of either ofthe transgenic PCR products and showing visible amounts of theendogenous PCR product, indicate that the corresponding plant from whichthe genomic template DNA was prepared, does not comprise the eliteevent. Lanes not showing visible amounts of the endogenous andtransgenic PCR products, indicate that the quality and/or quantity ofthe genomic DNA didn't allow for a PCR product to be generated. Theseplants cannot be scored. The genomic DNA preparation should be repeatedand a new PCR run, with the appropriate controls, has to be performed.

6.8. Use of Discriminating PCR Protocol to Identify EE-GM2

Before attempting to screen unknowns, a test run, with all appropriatecontrols, has to be performed. The developed protocol might requireoptimization for components that may differ between labs (template DNApreparation, Taq DNA polymerase, quality of the primers, dNTP's,thermocyler, etc.).

Amplification of the endogenous sequence plays a key role in theprotocol. One has to attain PCR and thermocycling conditions thatamplify equimolar quantities of both the endogenous and transgenicsequence in a known transgenic genomic DNA template. Whenever thetargeted endogenous fragment is not amplified or whenever the targetedsequences are not amplified with the same ethidium bromide stainingintensities, as judged by agarose gel electrophoresis, optimization ofthe PCR conditions may be required.

Glycine max leaf material from a number of plants, some of whichcomprising EE-GM2 were tested according to the above-described protocol.Samples from elite event EE-GM2 and from Glycine max wild-type weretaken as positive and negative controls, respectively.

FIG. 6 illustrates the result obtained with the elite event PCRIdentification Protocol for EE-GM2 on a number of soybean plant samples(lanes 1 to 14). The samples in lane 1 were found to contain the eliteevent as the 185 bp band is detected, while the samples in lanes 2, 3and 4 do not comprise EE-GM2. Lane 2 comprises another soybean eliteevent, lane 3 represents a non-transgenic Glycine max control; lane 4represents the negative control (water) sample, and lane 5 representsthe Molecular Weight Marker (100 bp).

7. Use of a Specific Integration Fragment as a Probe for Detection ofMaterial Comprising EE-GM2.

A specific integration fragment of EE-GM2 is obtained by PCRamplification using specific primers SOY09 (SEQ ID No. 18) and SOY010(SEQ ID No. 19) or by chemical synthesis and is labeled. Thisintegration fragment is used as a specific probe for the detection ofEE-GM2 in biological samples. Nucleic acid is extracted from the samplesaccording to standard procedures. This nucleic acid is then contactedwith the specific probe under hybridization conditions which areoptimized to allow formation of a hybrid. The formation of the hybrid isthen detected to indicate the presence of EE-GM2 nucleic acid in thesample. Optionally, the nucleic acid in the samples is amplified usingthe specific primers prior to contact with the specific probe.Alternatively, the nucleic acid is labeled prior to contact with thespecific probe instead of the integration fragment. Optionally, thespecific probe is attached to a solid carrier (such as, but not limitedto a filter, strip or beads), prior to contact with the samples.

8. Generation of Soybean Plants Comprising EE-GM3 and EE-GM1 or EE-GM3and EE-GM2 and Assessment of Agronomic Performance of Such Plants.

A soybean plant containing a combination of elite events EE-GM3 andEE-GM1 has been obtained by conventional crossing between a parentsoybean plant comprising EE-GM3 and a parent soybean plant comprisingEE-GM1. Progeny plants comprising both events are identified using PCRIdentification Protocols for EE-GM1 and EE-GM3 as described herein.Specifically, crosses were made with plants containing EE-GM3 andseveral EE-GM1 lines. Resulting F1 plants were grown and sprayed withglyphosate and glufosinate. F2 seed was harvested from F1 plants andplanted (F2 plants were not sprayed). F2 single plants were pulled.F2:F3 plant rows were grown and sprayed with glyphosate and glufosinate.Rows homozygous for both EE-GM3 and EE-GM1 were identified and laterharvested. Segregation data from this trial showed an expected Mendeliansegregation of the events.

A soybean plant containing a combination of elite events EE-GM3 andEE-GM2 has been obtained by conventional crossing between a parentsoybean plant comprising EE-GM3 and a parent soybean plant comprisingEE-GM2. Progeny plants comprising both events are identified using PCRIdentification Protocols for EE-GM2 and EE-GM3 as described herein.Specifically, crosses were made with plants containing EE-GM3 and plantscontaining EE-GM2. The resulting F1 plants were crossed to 4conventional lines. The F1s from these crosses were grown in the fieldand sprayed with glyphosate and glufosinate. F2 seed harvested from F1plants resistant to both herbicides was then planted. The F2 plants weresprayed with both glyphosate and glufosinate. Nine hundred plantstolerant to both herbicides were harvested and planted in a field trial.Expected Mendelian segregation data for the events was obtained in thistrial. Approximately 40 lines homozygous for tolerance to bothherbicides were selected for agronomic uniformity for further studies.These lines had good tolerance to both herbicides with good agronomiccharacteristics.

Field trials with such soybean plants comprising the combined events indifferent types of commercial germplasm are being conducted at multiplelocations and various agronomic parameters of the plants are beingevaluated, including plant height, height to node, stand, vigor, seedyield, and glyphosate, isoxaflutole and glufosinate tolerance levels.

9. Introgression of EE-GM3 and EE-GM1 or EE-GM2 into PreferredCultivars.

Elite event EE-GM3 and elite event EE-GM1 or EE-GM2 are introduced byrepeated back-crossing into commercial soybean cultivars such as but notlimited to Soybean Cultivar 7631014 (US2009252860); Soybean Cultivar7431014 (US2009252859); Soybean Cultivar 7925084 (US2009252858); SoybeanCultivar 7311153 (US2009252857); Soybean Cultivar S070159(US2009252856); Soybean Cultivar 7535357 (US2009246353); SoybeanCultivar S070160 (US2009246352); Soybean Cultivar 26074414(US2009249508); Soybean Cultivar 7509171 (US2009249507); SoybeanCultivar S070158 (US2009246351); Soybean Cultivar 7511119(US2009249506); Soybean Cultivar 7113111 (US2009238945); Soybeancultivar S06-02RM018047 (U.S. Pat. No. 7,592,518); Soybean Cultivar7013345 (US2009232957); Soybean Cultivar 7041461 (US2009235376); SoybeanCultivar 7549450 (US2009232956); Soybean Cultivar 7317090(US2009232955); Soybean Cultivar 2N2V58015 (US2009226597); SoybeanCultivar 7243182 (US2009226596); Soybean Cultivar 7143182(US2009226595); Soybean Cultivar 7043182 (US2009220673); SoybeanCultivar S070157 (US2009222950); Soybean Cultivar 306924721(US2009220672); Soybean Cultivar 7614385 (US2009220671); SoybeanCultivar 7925118 (US2009214750); Soybean Cultivar 7821295(US2009214749); Soybean Cultivar 7811336 (US2009214748); SoybeanCultivar S070150 (US2009214747); Soybean Cultivar 6214260(US2009214746); Soybean Cultivar S070152 (US2009214745); SoybeanCultivar 7429331 (US2009214751); Soybean Cultivar 26034631(US2009208634); Soybean cultivar 507-03JR108674 (U.S. Pat. No.7,560,621); Soybean cultivar 507-03KL016279 (U.S. Pat. No. 7,560,620);Soybean cultivar S06-CL959411 (U.S. Pat. No. 7,554,017); SOYBEANCULTIVAR SG3870NRR (US2009158453); SOYBEAN CULTIVAR HFPR-G (CA2645702);Soybean cultivar S06-02JR423016 (U.S. Pat. No. 7,521,606); Soybeancultivar S06-01JR119814 (U.S. Pat. No. 7,518,039); Soybean cultivarS06-01JR119448 (U.S. Pat. No. 7,518,038); Soybean Cultivar 6540220(US2009055960); Soybean Cultivar S060292 (US2009055959); SoybeanCultivar S050228 (US2009055958); Soybean cultivar S06-02JR423003 (U.S.Pat. No. 7,491,873); Soybean cultivar S06-02JR423005 (U.S. Pat. No.7,491,872); Soybean cultivar S06-02JR409114 (U.S. Pat. No. 7,485,782);Soybean cultivar S06-SJ144056 (U.S. Pat. No. 7,473,823); Soybeancultivar (U.S. Pat. No. 7,470,835); Soybean cultivar 6910450(US2008282369); SOYBEAN CULTIVAR 6223012 (U.S. Pat. No. 7,446,246);SOYBEAN CULTIVAR 6549250 (U.S. Pat. No. 7,446,245); Soybean Cultivar17731225 (US2008271204); Soybean Cultivar 6928285 (US2008271203);Soybean Cultivar 6736054 (US2008271169); Soybean Cultivar S060299(US2008271199); Soybean Cultivar S060294 (US2008271202); SoybeanCultivar 6943322 (US2008271201); Soybean cultivar 5343260(US2008263719); Soybean cultivar 6439359 (US2008263704); Soybeancultivar 6238359 (US2008263703); Soybean cultivar 6547272(US2008263702); Soybean cultivar 6929431 (US2008263701); Soybeancultivar 6703392 (US2008263700); Soybean cultivar 6044483(US2008263699); Soybean cultivar S050224 (US2008263698); Soybeancultivar 6719022 (US2008263697); Soybean cultivar 5826056(US2008263696); Soybean cultivar 6265047 (US2008263724); Soybeancultivar 6928331 (US2008263695); Soybean cultivar 6719331(US2008263694); Soybean cultivar 6636454 (US2008263693); Soybeancultivar 6226454 (US2008263718); Soybean cultivar Q0073801(US2008256657); SOYBEAN CULTIVAR 6326393 (US2008256652); SOYBEANCULTIVAR 6408448 (US2008256651); Soybean cultivar 6449315(US2008250524); Soybean cultivar S060296 (US2008250523); Soybeancultivar 6012078 (US2008250522); Soybean cultivar 6342078(US2008250521); Soybean cultivar 6419156 (US2008250520); Soybeancultivar 5723264 (US2008250519); Soybean cultivar S050225(US2008250518); Soybean cultivar S060298 (US2008244783); Soybeancultivar 6935331 (US2008244782); Soybean cultivar 6819456(US2008244787); Soybean cultivar S060297 (US2008244781); Soybeancultivar 6135319 (US2008244786); Soybean cultivar 6819331(US2008244780); Soybean cultivar 6137445 (US2008244779); Soybeancultivar 6917445 (US2008244778); Soybean cultivar 6111333(US2008244777); Soybean Cultivar S050229 (US2008244776); SoybeanCultivar 6114011 (US2008244775); Soybean Cultivar 6900358(US2008244784); Soybean Cultivar 6345184 (US2008244774); SoybeanCultivar 6836085 (US2008244773); Soybean Cultivar 6635047(US2008244772); Soybean Cultivar 6139105 (US2008244771); SOYBEANCULTIVAR 6434385 (US2008244770); SOYBEAN CULTIVAR S060295(US2008244769); Soybean Cultivar 6035184 (US2008244768); SoybeanCultivar S060293 (US2008209590); Soybean Cultivar 6733322(US2008209594); SOYBEAN CULTIVAR 6421326 (US2008209593); SoybeanCultivar S060077 (US2008209589); SOYBEAN CULTIVAR 6600375(US2008209592); Soybean cultivar S06-CL821457 (U.S. Pat. No. 7,420,104);Soybean cultivar S07-02KG294306 (U.S. Pat. No. 7,414,178); Soybeancultivar S06-BA046119 (U.S. Pat. No. 7,414,175); Soybean cultivarS07-02KG294307 (U.S. Pat. No. 7,411,114); Soybean Cultivar SG3865N(US2008189802); Soybean cultivar 6701475 (U.S. Pat. No. 7,408,097);Soybean Cultivar 1335025 (US2008178316); Soybean Cultivar 1686017(US2008178315); Soybean Cultivar 2388028 (US2008178314); SoybeanCultivar 2387029 (US2008178313); Soybean cultivar S06-WW152330 (U.S.Pat. No. 7,388,129); Soybean cultivar 6424090 (U.S. Pat. No. 7,385,118);Soybean cultivar 6723322 (U.S. Pat. No. 7,385,115); Soybean cultivarSG4377NRR (U.S. Pat. No. 7,385,114); Soybean cultivar S06-02JR111334(U.S. Pat. No. 7,381,864); Soybean cultivar 6141287 (U.S. Pat. No.7,378,577); Soybean cultivar MT110501 (U.S. Pat. No. 7,378,576); Soybeancultivar (U.S. Pat. No. 7,378,575); Soybean cultivar S06-WW169267 (U.S.Pat. No. 7,375,261); Soybean cultivar 6223392 (U.S. Pat. No. 7,371,939);Soybean cultivar S06-CL968413 (U.S. Pat. No. 7,371,937); Soybeancultivar S06-CL951107 (U.S. Pat. No. 7,368,636); Soybean cultivarS06-MT9152077 (U.S. Pat. No. 7,361,810); Soybean Cultivar 4211676(US2008092253); Soybean cultivar S06-M059029 (U.S. Pat. No. 7,355,101);Soybean Cultivar 6548193 (U.S. Pat. No. 7,345,228); Soybean cultivar6440261 (U.S. Pat. No. 7,345,227); Soybean cultivar S060291 (U.S. Pat.No. 7,342,151); Soybean cultivar S06-MT9206166 (U.S. Pat. No.7,339,094); Soybean cultivar S06-WW013107 (U.S. Pat. No. 7,339,093);Soybean cultivar S06-M03256 (U.S. Pat. No. 7,335,820); Soybean cultivar6134466 (U.S. Pat. No. 7,332,656); Soybean cultivar S06-01JR123373 (U.S.Pat. No. 7,329,800); Soybean cultivar S06-MT9211059 (U.S. Pat. No.7,326,831); Soybean cultivar 26170838 (US2008016590); Soybean cultivar306612412 (US2008016588); Soybean cultivar 26660135 (US2008016587);Soybean cultivar 306734323 (US2008016586); Soybean cultivarS06-01JR122235 (U.S. Pat. No. 7,317,144); SOYBEAN CULTIVAR 5900450 (U.S.Pat. No. 7,314,986); Soybean cultivar S06-MT116260 (U.S. Pat. No.7,314,984); Soybean cultivar S06-SJ143606 (U.S. Pat. No. 7,312,381);Soybean cultivar S06-98181-G01-35167 (U.S. Pat. No. 7,309,819); SOYBEANCULTIVAR 26082635 (U.S. Pat. No. 7,304,219); Soybean cultivar BA922834(U.S. Pat. No. 7,304,217); Soybean cultivar 01JR123480 (U.S. Pat. No.7,304,216); Soybean cultivar M061422 (U.S. Pat. No. 7,304,215); Soybeancultivar 17082821 (US2007277262); Soybean cultivar 17621620(US2007277261); Soybean cultivar 00977706 (US2007277260); Soybeancultivar S060182 (US2007277259); Soybean cultivar 26312034 (U.S. Pat.No. 7,301,078); Soybean cultivar 26143837 (U.S. Pat. No. 7,301,077);Soybean cultivar 435.TCS (US2007271626); Soybean cultivar 495.RC(US2007271625); Soybean cultivar 5306230 (U.S. Pat. No. 7,297,845);Soybean cultivar S06-WW167686 (U.S. Pat. No. 7,291,772); Soybeancultivar 6141145 (US2007245426); Soybean cultivar S050232(US2007226829); Soybean cultivar 5333301 (US2007226828); SOYBEANCULTIVAR S050215 (US2007226827); SOYBEAN CULTIVAR 3235020(US2007226826); Soybean cultivar 5720482 (US2007226825); Soybeancultivar S050216 (US2007226824); Soybean Cultivar 5512112(US2007226823); Soybean cultivar 3233021 (US2007226822); SOYBEANCULTIVAR 1336024 (US2007226821); Soybean cultivar 5348287(US2007226820); Soybean cultivar 5204220 (US2007226819); Soybeancultivar 6188027 (US2007226818); Soybean cultivar 4183026(US2007226817); Soybean cultivar S06-WW157958 (U.S. Pat. No. 7,271,325);Soybean cultivar 5733056 (US2007209091); Soybean cultivar 90501911(US2007209090); Soybean cultivar S050221 (US2007204361); SOYBEANCULTIVAR 5802205 (US2007204360); Soybean cultivar 1000642(US2007204359); Soybean cultivar 5420128 (US2007204358); Soybeancultivar S050222 (US2007199094); Soybean cultivar S050217(US2007199093); SOYBEAN CULTIVAR S050223 (US2007199092); Soybeancultivar S050218 (US2007199091); Soybean cultivar 5419227(US2007199089); Soybean cultivar 5319227 (US2007199088); Soybeancultivar 5723045 (US2007199087); SOYBEAN CULTIVAR 5051007(US2007199086); Soybean cultivar 5826175 (US2007192893); Soybeancultivar S050231 (US2007192892); SOYBEAN CULTIVAR 5826376(US2007192891); SOYBEAN CULTIVAR 5628386 (US2007192890); Soybeancultivar 5138236 (US2007186307); Soybean cultivar 5608398(US2007186306); SOYBEAN CULTIVAR S050230 (US2007186305); SOYBEANCULTIVAR 5624126 (US2007180561); SOYBEAN CULTIVAR 5019225(US2007180560); SOYBEAN CULTIVAR 5549483 (US2007180559); SOYBEANCULTIVAR 4189010 (US2007180551); SOYBEAN CULTIVAR 1486018(US2007180550); SOYBEAN CULTIVAR S050235 (US2007180549); SOYBEANCULTIVAR 5023230 (US2007180548); SOYBEAN CULTIVAR S050238(US2007174930); SOYBEAN CULTIVAR 5830261 (US2007174928); SOYBEANCULTIVAR S050226 (U.S. Pat. No. 7,247,772); SOYBEAN CULTIVAR 5806063(U.S. Pat. No. 7,247,771); SOYBEAN CULTIVAR S050233 (U.S. Pat. No.7,244,881); SOYBEAN CULTIVAR 5726085 (U.S. Pat. No. 7,241,939); Soybeancultivar MT000792 (U.S. Pat. No. 7,238,867); Soybean cultivar 5521161(U.S. Pat. No. 7,235,718); Soybean cultivar WW109447 (U.S. Pat. No.7,235,717); Soybean cultivar BA947474 (U.S. Pat. No. 7,220,898); Soybeancultivar 5939002 (U.S. Pat. No. 7,217,870); Soybean cultivar 5726175(U.S. Pat. No. 7,217,869); Soybean cultivar 5432082 (U.S. Pat. No.7,217,868); Soybean cultivar SG0850RR (U.S. Pat. No. 7,211,715); Soybeancultivar SG1750NRR (U.S. Pat. No. 7,208,658); Soybean cultivar MT017827(U.S. Pat. No. 7,208,657); Soybean cultivar 4N2V74028 (U.S. Pat. No.7,205,458); Soybean cultivar CL431203 (U.S. Pat. No. 7,202,400); Soybeancultivar 4N0S63222 (U.S. Pat. No. 7,199,288); Soybean cultivar 5520279(U.S. Pat. No. 7,196,253); Soybean cultivar 5834401 (U.S. Pat. No.7,196,252); Soybean cultivar 5621161 (U.S. Pat. No. 7,196,251); Soybeancultivar CL722114 (U.S. Pat. No. 7,196,250); Soybean cultivar 5741081(U.S. Pat. No. 7,193,140); Soybean cultivar CL727422 (U.S. Pat. No.7,186,895); Soybean cultivar 4N2V55022 (U.S. Pat. No. 7,183,468);Soybean cultivar 5083011 (U.S. Pat. No. 7,173,169); Soybean cultivar5626085 (U.S. Pat. No. 7,169,976); SOYBEAN CULTIVAR S050051 (U.S. Pat.No. 7,169,974); SOYBEAN CULTIVAR 4506816 (US2006294626); Soybeancultivar WW152201 (U.S. Pat. No. 7,132,594); Soybean cultivar CL727636(U.S. Pat. No. 7,132,593); Soybean cultivar M08851 (U.S. Pat. No.7,126,047); Soybean cultivar 4324401 (U.S. Pat. No. 7,105,728); Soybeancultivar S050164 (U.S. Pat. No. 7,105,727); Soybean cultivar 4136015(US2006195931); Soybean cultivar 3133014 (US2006195930); Soybeancultivar S040132 (US2006195929); Soybean Cultivar 4328386(US2006195928); Soybean cultivar 1339013 (US2006195927); SOYBEANCULTIVAR 4423183 (US2006195925); Soybean cultivar S040131(US2006195924); Soybean cultivar 4929388 (US2006195923); Soybeancultivar 4817034 (US2006195922); Soybean cultivar 4916816 (U.S. Pat. No.7,098,385); Soybean cultivar 4713487 (US2006191032); Soybean cultivar4348019 (US2006191031); Soybean cultivar S040122 (US2006191030); Soybeancultivar S040133 (US2006185031); Soybean cultivar CL821418 (U.S. Pat.No. 7,091,404); SOYBEAN CULTIVAR 4441080 (U.S. Pat. No. 7,091,403);Soybean cultivar 4805442 (US2006179509); Soybean cultivar 4921237(US2006179508); Soybean cultivar 4417380 (US2006174369); Soybeancultivar 4405070 (US2006174368); Soybean cultivar 4417779 (U.S. Pat. No.7,084,328); Soybean cultivar S040125 (US2006168678); Soybean cultivar4909380 (U.S. Pat. No. 7,081,572); Soybean cultivar S050162 (U.S. Pat.No. 7,081,571); Soybean cultivar 6084016 (U.S. Pat. No. 7,081,570);Soybean cultivar S050163 (U.S. Pat. No. 7,078,600); Soybean cultivarS040135 (U.S. Pat. No. 7,078,598); Soybean cultivar S040117 (U.S. Pat.No. 7,078,597); Soybean cultivar M03393 (U.S. Pat. No. 7,071,391);Soybean cultivar 4145306 (U.S. Pat. No. 7,064,253); Soybean cultivar900213 (US2006117405); Soybean cultivar 1000126 (US2006117404); Soybeancultivar 901023 (US2006117403); Soybean cultivar S040130 (U.S. Pat. No.7,053,280); Soybean cultivar 4706198 (U.S. Pat. No. 7,053,279); Soybeancultivar S040118 (U.S. Pat. No. 7,053,278); Soybean cultivar S040119(U.S. Pat. No. 7,053,277); Soybean cultivar S040123 (U.S. Pat. No.7,053,276); Soybean cultivar 4442112 (U.S. Pat. No. 7,049,497); SOYBEANCULTIVAR 917013 (U.S. Pat. No. 7,045,689); Soybean cultivar S040124(U.S. Pat. No. 7,045,691); Soybean cultivar 4238491 (U.S. Pat. No.7,045,690); Soybean cultivar S010136 (U.S. Pat. No. 7,041,882); Soybeancultivar 900613 (U.S. Pat. No. 7,030,297); Soybean cultivar 4337175(U.S. Pat. No. 7,030,301); Soybean cultivar S040121 (U.S. Pat. No.7,030,300); Soybean cultivar 4216033 (U.S. Pat. No. 7,030,299); Soybeancultivar S040128 (U.S. Pat. No. 7,022,901); Soybean cultivar S040120(U.S. Pat. No. 7,022,900); Soybean cultivar S040127 (U.S. Pat. No.7,019,199); Soybean cultivar S040134 (U.S. Pat. No. 7,015,378); Soybeancultivar S040129 (U.S. Pat. No. 7,015,377); Soybean cultivar 4513420(U.S. Pat. No. 7,005,564); Soybean cultivar 943013 (US2006031958);Soybean cultivar S030136 (US2006021081); Soybean cultivar 927013(US2006021080); Soybean cultivar 1000109 (US2006015962); Soybeancultivar 90046112 (US2006010530); Soybean cultivar 90897327(US2006010529); Soybean cultivar 90362421 (US2006010528); Soybeancultivar 03022253 (US2006010527); Soybean cultivar 02022433(US2006010526); Soybean cultivar 02022323 (US2006010525); Soybeancultivar 02912951 (US2006010524); Soybean cultivar 0102115(US2006010523); Soybean cultivar 915034 (US2006010522); Soybean cultivar0509255 (US2006010521); Soybean cultivar 4803070 (U.S. Pat. No.6,982,368); Soybean cultivar 4704310 (U.S. Pat. No. 6,979,762); Soybeancultivar SJ919784 (US2005268362); Soybean cultivar CL615261(US2005268361); Novel soybean (US2004199964); Soybean cultivar 0509214(US2005210542); Soybean cultivar 70826751 (US2005193442); Soybeancultivar 0509243 (US2005193441); Soybean cultivar 0509246(US2005193440); Soybean cultivar 0509253 (US2005193439); Soybeancultivar 0509247 (US2005193438); Soybean cultivar 0509252(US2005193437); Soybean cultivar 0509241 (US2005193436); Soybeancultivar 0509249 (U.S. Pat. No. 6,884,927); Soybean cultivar 0509244(US2005183158); Soybean cultivar 0509240 (US2005183157); Soybeancultivar 0509239 (US2005183156); Soybean cultivar 0509254(US2005183155); Soybean cultivar 0509245 (US2005183154); Soybeancultivar 0509251 (US2005183153); Soybean cultivar 4283008 (U.S. Pat. No.6,888,050); Soybean cultivar 2386009 (US2005183152); Soybean cultivar3282002 (U.S. Pat. No. 6,870,080); Soybean cultivar 0509248(US2005183151); Soybean cultivar 5091007 (U.S. Pat. No. 6,906,249);Soybean cultivar 0509236 (US2005166281); Soybean cultivar 0509235(US2005166280); Soybean cultivar 0509237 (US2005166279); Soybeancultivar SG5322NRR (US2005164390); Soybean cultivar SG5030NRR(US2005166278); Soybean cultivar SG4911NRR (US2005166277); Soybeancultivar S030153 (US2005160504); Soybean cultivar S030158(US2005144680); SOYBEAN CULTIVAR S030160 (US2005144679); Soybeancultivar S030161 (US2005144678); Soybean cultivar S030157(US2005144677); Soybean cultivar S030155 (US2005144676); Soybeancultivar S030156 (US2005144675); SOYBEAN CULTIVAR S030159(US2005144674); Soybean cultivar S030154 (U.S. Pat. No. 6,900,376);Soybean cultivar S020030 (US2005114929); Soybean cultivar S030010(US2005114928); Soybean cultivar SG1431RR (US2005097629); SOYBEANCULTIVAR SG1330NRR (US2005097642); Soybean cultivar S030150(US2005071900); SOYBEAN CULTIVAR S022209 (US2005050601); Soybeancultivar S022217 (US2005050600); Soybean cultivar S022219(US2005050599); Soybean cultivar S030151 (US2005050598); Soybeancultivar 0491735 (US2005022272); Soybean cultivar SO22218(US2005022271); Soybean cultivar 6190006 (US2004268447); Soybeancultivar SG1120RR (US2004250316); Soybean cultivar 0487681(US2004237153); Soybean cultivar 0491717 (US2004237152); Soybeancultivar SO22220 (US2004237151); Soybean cultivar 0491715(US2004237150); Soybean cultivar 0491712 (US2004237149); Soybeancultivar 0491718 (US2004237148); Soybean cultivar 99271316(US2004221344); Soybean cultivar SO22212 (US2004221343); Soybeancultivar 0491737 (US2004221342); Soybean cultivar SO22211(US2004221341); Soybean cultivar SO22210 (US2004221340); Soybeancultivar SO22213 (US2004221339); Soybean cultivar 0491725(US2004221346); Soybean cultivar 03129016 (US2004221329); Soybeancultivar SO22208 (US2004221328); Soybean cultivar SO22207(US2004221345); Soybean cultivar 02932 (US2004210968); Soybean cultivar94137321 (US2004205862); Soybean cultivar 94106224 (US2004205861);Soybean cultivar 94143901 (US2004205859); SOYBEAN CULTIVAR 0487685(US2004205858); SOYBEAN CULTIVAR 92440927 (US2004205857); Soybeancultivar 0487686 (US2004205856); Soybean cultivar SO22215(US2004205855); Soybean cultivar SO22214 (US2004205854); SOYBEANCULTIVAR 0491726 (US2004205849); SOYBEAN CULTIVAR 92478609(US2004205853); Soybean cultivar 922013 (U.S. Pat. No. 6,781,040);SOYBEAN CULTIVAR 0491727 (US2004205852); SOYBEAN CULTIVAR 0491728(US2004205851); Soybean cultivar 1465003 (US2004098766); Soybeancultivar 3186004 (US2004019936); Soybean cultivar 7085005(US2004205850); Soybean cultivar SO22204 (US2004199958); Soybeancultivar SO22206 (US2004199957); Soybean cultivar 0491731(US2004199956); Soybean cultivar 0491733 (US2004199955); Soybeancultivar 0491738 (US2004199954); Soybean cultivar 0491732(US2004199953); Soybean cultivar 0491729 (US2004199952); Soybeancultivar SO20011 (US2004199951); Soybean cultivar 0491739(US2004199950); Soybean cultivar 0491730 (US2004199949); Soybeancultivar 13873 (US2004199948); Soybean cultivar 954011 (US2004181822);Soybean cultivar 010022 (US2004181831); Soybean cultivar 4181001(US2003229926); Soybean cultivar 0491721 (US2004168228); Soybeancultivar 0491723 (U.S. Pat. No. 6,911,581); Soybean cultivar 0491716(US2004168226); Soybean cultivar 0491713 (US2004168225); Soybeancultivar 0491711 (US2004168224); Soybean cultivar 0491722(US2004168223); Soybean cultivar 0491724 (US2004168222); Soybeancultivar 0491720 (US2004168221); Soybean cultivar 0487682(US2004168220); Soybean cultivar 0491714 (US2004168219); Soybeancultivar 0491719 (US2004168218); Soybean cultivar DP 5634 RR(US2003177540); Soybean Cultivar S56-D7 (US2004098765); Soybean cultivar926877 (US2004064859); Soybean cultivar SE73753 (US2004055059); Soybeancultivar SN83594 (US2004055058); Soybean cultivar SE71112(US2004055056); Soybean cultivar SE73090 (US2004055054); Soybeancultivar SN79526 (US2004055053); Soybean cultivar SW90702(US2004055052); Soybean cultivar SN79525 (US2004055051); Soybeancultivar SE90345 (US2004055050); Soybean cultivar 0149928(US2004055049); Soybean cultivar SN83780 (US2004055048); Soybeancultivar 0053840 (US2004055047); Soybean cultivar 924001 (US2004055046);Soybean cultivar 0004747 (US2004055057); Soybean cultivar 0037357(US2004055045); Soybean cultivar SN83366 (US2004055044); Soybeancultivar SN76208 (US2004055043); Soybean cultivar 0037370(US2004055042); Soybean cultivar SX95512 (US2004049821); Soybeancultivar 0096008 (US2004049820); Soybean cultivar SN83544(US2004049819); Soybean cultivar 0088401 (US2004049818); Soybeancultivar SN64195 (US2004049817); Soybean cultivar 0034754(US2004049816); Soybean cultivar SN71173 (US2004049815); Soybeancultivar SN83211 (US2004049814); Soybean cultivar 92422749(US2004045058); Soybean cultivar 0120311 (US2004045057); Soybeancultivar S010344 (US2004003438); Soybean cultivar 70876922(US2004003437); Soybean cultivar 924496 (US2004003436); Soybean cultivar19705120 (US2003237116); Soybean cultivar 19704220 (US2003235914);Soybean Cultivar 19704280 (US2003237115); Soybean cultivar 19704210(US2003237114); Soybean cultivar S37-N4 (US2003237113); Soybean cultivar19602310 (US2003233685); Soybean cultivar 19704120 (US2003233684);Soybean cultivar 19704230 (US2003233683); Soybean cultivar 1000126(US2003233682); Soybean cultivar 93831526 (US2003221226); Soybeancultivar 0322581 (US2003221225); Soybean cultivar 0332149(US2003213028); Soybean cultivar 0332144 (US2003213027); Soybeancultivar 924788 (US2003213026); Soybean cultivar 924861 (US2003213025);Soybean cultivar 928070 (US2003213024); Soybean cultivar S010354(US2003213023); Soybean cultivar S010360 (US2003213022); Soybeancultivar S010361 (US2003213021); Soybean cultivar S010363(US2003213020); Soybean cultivar S010364 (US2003213019); Soybeancultivar 0332148 (US2003208805); Soybean cultivar 0332147(US2003208804); Soybean cultivar 0332146 (US2003208803); Soybeancultivar 0332135 (US2003208802); Soybean cultivar 1000144(US2003208801); Soybean cultivar 0332143 (US2003208800); Soybeancultivar 0332145 (US2003208799); Soybean cultivar S010345(US2003204884); Soybean cultivar 0332131 (US2003204883); Soybeancultivar 0332130 (US2003204882); Soybean cultivar 0332129(US2003204881); Soybean cultivar 0332122 (US2003204880); Soybeancultivar S010350 (US2003204879); Soybean cultivar S010355(US2003204878); Soybean cultivar 031766 (US2003204877); Soybean cultivarS010353 (US2003204876); Soybean cultivar 0322580 (US2003200579); Soybeancultivar 0322579 (US2003200578); Soybean cultivar S010347(US2003200577); Soybean cultivar S010349 (US2003200576); Soybeancultivar 0332141 (US2003200575); Soybean cultivar 0332142(US2003200574); Soybean Cultivar 0332133 (US2003200573); Soybeancultivar 0332134 (US2003200572); Soybean cultivar 0332139(US2003200571); Soybean cultivar 0332137 (US2003200570); Soybean varietyXB33U08 (U.S. Pat. No. 7,598,435); Soybean variety XB27D08 (U.S. Pat.No. 7,592,519); Soybean variety XB41M08 (U.S. Pat. No. 7,589,261);Soybean variety XB05J08 (U.S. Pat. No. 7,589,260); Soybean varietyXB33T08 (U.S. Pat. No. 7,589,259); Soybean variety XB30Y08 (U.S. Pat.No. 7,586,025); Soybean variety XB40U08 (U.S. Pat. No. 7,582,813);Soybean variety XB29M08 (U.S. Pat. No. 7,582,811); SOYBEAN VARIETY 93Y10(US2009144843); SOYBEAN VARIETY D4325666 (US2009055957); SOYBEAN VARIETYD4125897 (US2009055956); SOYBEAN VARIETY D4698573 (US2009055955);SOYBEAN VARIETY D4356652 (US2009019592); SOYBEAN VARIETY D4456885(US2009019591); SOYBEAN VARIETY D4698013 (US2009019590); SOYBEAN VARIETYD4637114 (US2009019589); SOYBEAN VARIETY D4102367 (US2009019595);SOYBEAN VARIETY D4266582 (US2009019594); SOYBEAN VARIETY D4422801(US2009019593); SOYBEAN VARIETY D4520980 (US2009019588); SOYBEAN VARIETYD4521369 (US2009019587); SOYBEAN VARIETY D4223057 (US2009019586);SOYBEAN VARIETY D4682156 (US2009019585); SOYBEAN VARIETY D4233569(US2009019584); SOYBEAN VARIETY D4925614 (US2009019583); SOYBEAN VARIETYD4203144 (US2009019604); SOYBEAN VARIETY D4102536 (US2009019582);SOYBEAN VARIETY D4865324 (US2009019581); SOYBEAN VARIETY D4825495(US2009019580); SOYBEAN VARIETY D4659251 (US2009019579); SOYBEAN VARIETYD4258962 (US2009019578); SOYBEAN VARIETY D4253969 (US2009019577);SOYBEAN VARIETY D4696658 (US2009019603); SOYBEAN VARIETY D4256925(US2009019576); SOYBEAN VARIETY D4253681 (US2009019575); SOYBEAN VARIETYD4789254 (US2009019574); SOYBEAN VARIETY D4713125 (US2009019573);SOYBEAN VARIETY D4526223 (US2009019572); SOYBEAN VARIETY D4556201(US2009019571); SOYBEAN VARIETY D4012368 (US2009019570); SOYBEAN VARIETYD4452019 (US2009019569); SOYBEAN VARIETY D4201483 (US2009019568);SOYBEAN VARIETY D4463892 (US2009019567); SOYBEAN VARIETY D4159630(US2009019566); SOYBEAN VARIETY D4470236 (US2009019565); SOYBEAN VARIETYD4063284 (US2009019564); SOYBEAN VARIETY D4021792 (US2009013429);SOYBEAN VARIETY D4902530 (US2009013428); SOYBEAN VARIETY D4367012(US2009013427); SOYBEAN VARIETY D4923560 (US2009013426); SOYBEAN VARIETYD4253854 (US2009013425); SOYBEAN VARIETY D4210110 (US2009007290);SOYBEAN VARIETY D4523081 (US2009007289); SOYBEAN VARIETY D4328762(US2009007288); SOYBEAN VARIETY D4483789 (US2009007287); SOYBEAN VARIETYD4311702 (US2009007286); SOYBEAN VARIETY D4127789 (US2008313765);SOYBEAN VARIETY D4361423 (US2008313764); SOYBEAN VARIETY D4208814(US2008313763); SOYBEAN VARIETY D4201139 (US2008313762); SOYBEAN VARIETYD4120384 (US2008313761); SOYBEAN VARIETY D4572906 (US2008313760);SOYBEAN VARIETY D4301279 (US2008313759); SOYBEAN VARIETY D4422957(US2008313758); SOYBEAN VARIETY D4256958 (US2008313757); SOYBEAN VARIETY4074328 (US2008282366); SOYBEAN VARIETY XB47Q06 (US2008244767); SOYBEANVARIETY XB26R06 (US2008244766); SOYBEAN VARIETY 4991629 (US2008216190);SOYBEAN VARIETY 4158090 (US2008216189); Soybean Variety XB40K07(US2008209582); SOYBEAN VARIETY D0069201 (US2008178345); SOYBEAN VARIETYD0064801 (US2008178320); SOYBEAN VARIETY D0063801 (US2008178344);SOYBEAN VARIETY D0061501 (US2008178343); SOYBEAN VARIETY 4938051(US2008178319); SOYBEAN VARIETY 4880500 (US2008178318); SOYBEAN VARIETY4835953 (US2008178317); SOYBEAN VARIETY 4684181 (US2008178342); SOYBEANVARIETY 4427363 (US2008178340); SOYBEAN VARIETY 4676311 (US2008178339);SOYBEAN VARIETY 4953710 (US2008178337); SOYBEAN VARIETY 4857548(US2008178336); SOYBEAN VARIETY 4551757 (US2008178335); SOYBEAN VARIETY4027271 (US2008178334); SOYBEAN VARIETY 4274171 (US2008178333); SOYBEANVARIETY 0341931 (US2008178332); SOYBEAN VARIETY 4282159 (US2008178331);SOYBEAN VARIETY 4852004 (US2008178330); SOYBEAN VARIETY 4688589(US2008178329); SOYBEAN VARIETY 4614131 (US2008178327); SOYBEAN VARIETY4201823 (US2008178326); SOYBEAN VARIETY 92M22 (US2008178350); SOYBEANVARIETY 4174206 (US2008178322); SOYBEAN VARIETY 4305498 (US2008178321);SOYBEAN VARIETY 4423586 (US2008172761); SOYBEAN VARIETY 4568207(US2008172756); SOYBEAN VARIETY 4840308 (US2008172755); SOYBEAN VARIETY4256323 (US2008172754); SOYBEAN VARIETY 4789516 (U.S. Pat. No.7,399,907); SOYBEAN VARIETY 90Y40 (US2008168581); SOYBEAN VARIETY4959932 (U.S. Pat. No. 7,396,983); SOYBEAN VARIETY 4062885 (U.S. Pat.No. 7,394,000); Soybean variety 4858197 (U.S. Pat. No. 7,390,940);Soybean variety 4092390 (U.S. Pat. No. 7,390,939); Soybean variety4735486 (U.S. Pat. No. 7,390,938); Soybean variety 4219527 (U.S. Pat.No. 7,388,132); Soybean variety 4599695 (U.S. Pat. No. 7,388,131);Soybean variety 4554257 (U.S. Pat. No. 7,388,130); Soybean variety4896902 (U.S. Pat. No. 7,385,113); Soybean variety 4367308 (U.S. Pat.No. 7,385,112); Soybean variety 4589609 (U.S. Pat. No. 7,385,111);Soybean variety 4640250 (U.S. Pat. No. 7,385,110); Soybean variety4540394 (U.S. Pat. No. 7,385,109); Soybean variety 4297661 (U.S. Pat.No. 7,385,108); Soybean variety 4958786 (U.S. Pat. No. 7,381,866);Soybean variety 4440685 (U.S. Pat. No. 7,375,262); Soybean variety4008211 (U.S. Pat. No. 7,371,938); Soybean variety 4778469 (U.S. Pat.No. 7,368,637); Soybean variety 4766295 (U.S. Pat. No. 7,355,103);Soybean variety 4436909 (U.S. Pat. No. 7,355,102); Soybean variety4812469 (U.S. Pat. No. 7,351,886); Soybean variety 4761767 (U.S. Pat.No. 7,351,885); Soybean variety 4142393 (U.S. Pat. No. 7,329,801);Soybean variety 4502135 (U.S. Pat. No. 7,326,832); Soybean variety4353363 (U.S. Pat. No. 7,321,082); Soybean variety 91B42 (U.S. Pat. No.7,317,143); SOYBEAN VARIETY 0330739 (US2007271622); Soybean variety0384279 (U.S. Pat. No. 7,294,768); SOYBEAN VARIETY 4175567(US2007256187); SOYBEAN VARIETY 4336643 (US2007256186); SOYBEAN VARIETY4671685 (US2007256185); SOYBEAN VARIETY 4309194 (US2007256190); SOYBEANVARIETY 0330738 (US2007256184); SOYBEAN VARIETY 0045477 (US2007256183);SOYBEAN VARIETY 0437973 (US2007256182); SOYBEAN VARIETY 0457028(US2007256181); SOYBEAN VARIETY 0367478 (US2007256180); SOYBEAN VARIETY0358232 (US2007256179); SOYBEAN VARIETY 0417158 (US2007256178); SOYBEANVARIETY 4559809 (US2007256177); SOYBEAN VARIETY 0196172 (US2007256176);SOYBEAN VARIETY 4785923 (US2007256175); SOYBEAN VARIETY 4587513(US2007256174); SOYBEAN VARIETY 0409670 (US2007256173); SOYBEAN VARIETY4010165 (US2007256172); SOYBEAN VARIETY 0421133 (US2007256171); SOYBEANVARIETY 0240187 (US2007256170); SOYBEAN VARIETY 0387907 (US2007256169);SOYBEAN VARIETY 0232405 (US2007256168); SOYBEAN VARIETY 0146529(US2007256167); SOYBEAN VARIETY 4788561 (US2007256166); SOYBEAN VARIETY457114 (US2007256165); SOYBEAN VARIETY 0149217 (US2007256164); SOYBEANVARIETY 4247825 (US2007254366); SOYBEAN VARIETY 0212938 (US2007256163);SOYBEAN VARIETY 0146565 (US2007256162); SOYBEAN VARIETY 4647672(US2007256161); SOYBEAN VARIETY 0215818 (US2007256160); SOYBEAN VARIETY0384531 (US2007256159); SOYBEAN VARIETY 4878185 (US2007254365); SOYBEANVARIETY 4498438 (US2007256158); SOYBEAN VARIETY 0436052 (US2007256157);SOYBEAN VARIETY 4782157 (US2007256156); SOYBEAN VARIETY 0385457(US2007256155); SOYBEAN VARIETY 0385240 (US2007256154); SOYBEAN VARIETY4735316 (US2007256153); SOYBEAN VARIETY 0277524 (US2007256152); SOYBEANVARIETY 0276951 (US2007256151); Soybean Variety XB37L07 (US2007245429);Soybean Variety XB35X07 (US2007226837); Soybean Variety XB35S07(US2007226836); Soybean Variety XB35F07 (US2007226835); Soybean VarietyXB34R07 (US2007226834); Soybean Variety XB34L07 (US2007226833); SoybeanVariety XB34D07 (US2007226832); Soybean Variety XB33G07 (US2007226831);Soybean Variety 98Y11 (US2007169220); Soybean variety 0137335 (U.S. Pat.No. 7,241,941); Soybean Variety XB15E07 (US2007150980); Soybean Variety92M52 (US2007150979); Soybean Variety XB47R07 (US2007136888); SoybeanVariety XB46V07 (US2007136887); Soybean Variety XB57E07 (US2007136886);Soybean Variety XB54X07 (US2007136885); Soybean Variety XB54V07(US2007136884); Soybean Variety XB52Q07 (US2007136883); Soybean VarietyXB37M07 (US2007136882); Soybean Variety XB37J07 (US2007136881); SoybeanVariety XB34Q07 (US2007136880); Soybean Variety XB32S07 (US2007136879);Soybean Variety XB32J07 (US2007136878); Soybean Variety XB31R07(US2007136877); Soybean Variety XB31J07 (US2007136876); Soybean VarietyXB29K07 (US2007136875); Soybean Variety XB31H07 (US2007136874); SoybeanVariety XB30G07 (US2007136873); Soybean Variety XB30E07 (US2007136872);Soybean Variety XB25E07 (US2007136871); Soybean Variety XB26X07(US2007136870); Soybean Variety XB23L07 (US2007136869); Soybean VarietyXB19Z07 (US2007136868); Soybean Variety XB19E07 (US2007136867); SoybeanVariety XB18M07 (US2007136866); Soybean Variety XB18K07 (US2007136865);Soybean Variety XB18J07 (US2007136864); Soybean Variety XB17W07(US2007136863); Soybean Variety XB17U07 (US2007136862); Soybean VarietyXB15B07 (US2007136861); Soybean Variety XB12R07 (US2007136860); SoybeanVariety XB11J07 (US2007136859); Soybean Variety XB04E07 (US2007136858);Soybean Variety XB02K07 (US2007136857); Soybean Variety XB49V07(US2007136856); Soybean Variety XB48X07 (US2007136855); Soybean Variety92M75 (US2007136854); Soybean Variety XB48W07 (US2007136853); SoybeanVariety XB44G07 (US2007136852); Soybean Variety XB42K07 (US2007136851);Soybean Variety XB42H07 (US2007136850); Soybean Variety XB41J07(US2007136849); Soybean Variety XB40Y07 (US2007136848); Soybean VarietyXB40X07 (US2007136847); Soybean Variety XB39E07 (US2007136846); SoybeanVariety XB38W07 (US2007136845); Soybean Variety XB38S07 (US2007136844);Soybean Variety XB23V07 (US2007136843); Soybean Variety XB31M07(US2007130652); Soybean Variety XB28E07 (US2007130651); Soybean VarietyXB25S07 (US2007130650); Soybean Variety XB21N07 (US2007130649); SoybeanVariety XB03Q07 (US2007130648); Soybean Variety XB49Q07 (US2007130647);Soybean Variety XB06M07 (US2007130646); Soybean variety S04-97130-15-02(U.S. Pat. No. 7,196,249); Soybean variety S04-97026-N99-42648-01 (U.S.Pat. No. 7,189,896); Soybean variety S05-97016-G99-21212 (U.S. Pat. No.7,186,894); Soybean variety S05-99048-19 (U.S. Pat. No. 7,164,064);Soybean variety 92B14 (U.S. Pat. No. 7,161,065); Soybean Variety 98R31(US2007006350); Soybean variety 505-97177-N00-22972 (U.S. Pat. No.7,132,592); Soybean variety XB25G06 (US2006225160); Soybean variety91M70 (US2006174381); Soybean variety XB24R06 (US2006162029); Soybeanvariety S03-95368-N98-52902 (U.S. Pat. No. 7,078,594); Soybean varietyS05-97130-51 (U.S. Pat. No. 7,078,599); Soybean variety XB11L06(US2006130187); Soybean variety 94B13 (U.S. Pat. No. 7,064,251); Soybeanvariety 94B74 (U.S. Pat. No. 7,064,250); Soybean variety XB27J06(US2006112462); Soybean variety XB29N06 (US2006112460); Soybean varietyXB28T06 (US2006112459); Soybean variety XB16W06 (US2006112458); Soybeanvariety XB18C06 (US2006112456); Soybean variety XB 10M06 (US2006107391);Soybean variety XB06K06 (US2006107390); Soybean variety XB28V06(US2006107389); Soybean variety XB004A06 (US2006107388); Soybean varietyXB12L06 (US2006107387); Soybean variety XB005A06 (US2006107386); Soybeanvariety XB25H06 (US2006107385); Soybean variety XB39WO6 (US2006107384);Soybean variety XB27K06 (US2006107383); Soybean variety XB29R06(US2006107382); Soybean variety XB16S06 (US2006107381); Soybean varietyXB36V06 (US2006107380); Soybean variety XB07N06 (US2006107379); Soybeanvariety XB23H06 (US2006107378); Soybean variety XB35C06 (US2006107377);Soybean variety XB32L06 (US2006107376); Soybean variety XB58P06(US2006107375); Soybean variety XB36M06 (US2006107374); Soybean varietyXB22G06 (US2006107373); Soybean variety XB36Q06 (US2006107372); Soybeanvariety 91M61 (US2006107371); Soybean variety XB32A06 (US2006107370);Soybean variety XB19V06 (US2006107369); Soybean variety XB43C06(US2006107368); Soybean variety XB22N06 (US2006107367); Soybean varietyXB38E06 (US2006107366); Soybean variety XB37U06 (US2006107365); Soybeanvariety XB37Q06 (US2006107364); Soybean variety XB00D06 (US2006107363);Soybean variety XB14N06 (US2006107362); Soybean variety XB31H06(US2006107361); Soybean variety XB21Z06 (US2006107360); Soybean varietyXB005B06 (US2006107359); Soybean variety XB15W06 (US2006107358); Soybeanvariety XB33N06 (US2006107357); Soybean variety XB18W06 (US2006107356);Soybean variety XB32M06 (US2006107355); Soybean variety XB19F06(US2006107354); Soybean variety S03-95021-55-138-AB (U.S. Pat. No.7,026,531); Soybean variety 94M41 (U.S. Pat. No. 7,002,061); Soybeanvariety 91M50 (U.S. Pat. No. 6,998,518); Soybean variety 92B13 (U.S.Pat. No. 6,989,475); Soybean variety 93B68 (U.S. Pat. No. 6,989,474);Soybean variety 93B09 (U.S. Pat. No. 6,979,759); Soybean variety 92M00(U.S. Pat. No. 6,972,352); Soybean variety XB08P05 (US2005120433);Soybean variety XB26V05 (US2005150023); Soybean variety XB21R05(US2005108795); Soybean variety XB28E05 (US2005114942); Soybean varietyXB58K05 (US2005114941); Soybean variety XB27B05 (US2005114940); Soybeanvariety XB21S05 (US2005150022); Soybean variety XB26U05 (US2005138695);Soybean variety XB35K05 (US2005150021); Soybean variety XB18S05(US2005120436); Soybean variety XB25C05 (US2005120435); Soybean variety90M01 (US2005120434); Soybean variety XB22H05 (US2005150020); Soybeanvariety XB22K05 (US2005114939); Soybean variety XB58G05 (US2005114938);Soybean variety XB57U05 (US2005120432); Soybean variety XB49M05(US2005120431); Soybean variety XB20D05 (US2005144683); Soybean varietyXB41B05 (US2005150019); Soybean variety XB38T05 (US2005120430); Soybeanvariety XB13T05 (US2005120429); Soybean variety XB19Y05 (US2005120428);Soybean variety XB43D05 (US2005120427); Soybean variety XB40E05(US2005120426); Soybean variety XB39N05 (US2005120425); Soybean variety93M01 (US2005120424); SOYBEAN VARIETY XB31W05 (US2005223439); Soybeanvariety XB32C05 (US2005114937); Soybean variety XB40D05 (US2005120423);Soybean variety 92M61 (US2005120422); Soybean variety 91M91(US2005114936); Soybean variety XB33Y05 (US2005120421); Soybean varietyXB34A05 (US2005120420); Soybean variety XB13U05 (US2005114935); Soybeanvariety XB12K05 (US2005114934); Soybean variety XB30P05 (US2005120419);Soybean variety XB57T05 (US2005114933); Soybean variety XB17S05(US2005114932); Soybean variety XB25Y05 (US2005114930); Soybean varietyXB25S05 (US2005150017); Soybean variety XB43W04 (US2004177420); Soybeanvariety XB44W04 (US2004177419); Soybean variety XB53J04 (US2004199960);Soybean variety XB43V04 (US2004216192); Soybean variety XB49K04(US2004172668); Soybean variety XB27P04 (US2004205864); Soybean varietyXB29L04 (US2004177418); Soybean variety XB29K04 (US2004177417); Soybeanvariety XB41U04 (US2004231017); Soybean variety XB34D04 (US2004177416);Soybean variety XB09J04 (US2004172711); Soybean variety XB32Y04(US2004194169); Soybean variety XB44D04 (US2004172710); Soybean varietyXB44C04 (US2004172709); Soybean variety XB10L04 (US2004172708); Soybeanvariety XB19U04 (US2004172707); Soybean variety XB02F04 (US2004172706);Soybean variety XB25X04 (US2004172705); Soybean variety XB26L04(US2004172704); Soybean variety XB11F04 (US2004172703); Soybean varietyXB40Z04 (US2004177415); Soybean variety XB40Y04 (US2004181833); Soybeanvariety XB007C04 (US2004181832); Soybean variety 96M20 (US2004172702);Soybean variety XB39J04 (US2004172701); Soybean variety XB29A04(US2004172700); Soybean variety XB35P04 (US2004172699); Soybean varietyXB58Z04 (US2004177414); Soybean variety XB43R04 (US2004172698); Soybeanvariety XB35L04 (US2004172697); Soybean variety XB06H04 (US2004172696);Soybean variety XB59U04 (US2004172695); Soybean variety XB64C04(US2004172694); Soybean variety 95M80 (US2004172693); Soybean varietyXB35Q04 (US2004177413); Soybean variety XB04D04 (US2004177412); Soybeanvariety XB08L04 (US2004177411); Soybean variety XB18Q04 (US2004177410);Soybean variety XB16Q04 (US2004177409); Soybean variety XB55K04(US2004172692); Soybean variety XB57M04 (US2004172691); Soybean varietyXB25L04 (US2004205863); Soybean variety XB48T04 (US2004194168); Soybeanvariety XB42X04 (US2004199959); Soybean variety XB31T04 (US2004177408);Soybean variety XB31U04 (US2004194167); Soybean variety XB30E04(US2004177407); Soybean variety XB31R04 (US2004177406); Soybean varietyS03-95341-A98-60618 (U.S. Pat. No. 6,909,033); Soybean variety SN97-6946(US2004168227); Soybean variety 94M70 (U.S. Pat. No. 6,864,408); Soybeanvariety 92M70 (U.S. Pat. No. 6,797,866); Soybean variety 92M71 (U.S.Pat. No. 6,858,782); Soybean variety 91M40 (U.S. Pat. No. 6,828,490);Soybean variety 93M80 (U.S. Pat. No. 6,849,789); Soybean variety XB39N03(U.S. Pat. No. 6,864,407); Soybean variety 93M90 (U.S. Pat. No.6,846,975); Soybean variety 90M90 (U.S. Pat. No. 6,852,913); Soybeanvariety 92M72 (U.S. Pat. No. 6,960,708); Soybean variety 91M90 (U.S.Pat. No. 6,849,788); Soybean variety 92M50 (U.S. Pat. No. 6,855,876);Soybean variety 92M30 (U.S. Pat. No. 6,951,974); Soybean variety 93M60(U.S. Pat. No. 6,797,865); Soybean variety 93M40 (U.S. Pat. No.6,791,016); Soybean variety 93M41 (U.S. Pat. No. 6,835,875); Soybeanvariety XB15P03 (U.S. Pat. No. 6,797,864); Soybean variety XB24H03 (U.S.Pat. No. 6,936,752); Soybean variety XB05A03 (U.S. Pat. No. 6,815,585);Soybean variety 92M80 (U.S. Pat. No. 6,849,787); Soybean variety XB33S03(U.S. Pat. No. 6,855,875); Soybean variety XB48P03 (U.S. Pat. No.6,797,863); Soybean variety XB29X03 (U.S. Pat. No. 6,806,406); Soybeanvariety XB02C03 (U.S. Pat. No. 6,800,795); Soybean variety XB29W03 (U.S.Pat. No. 6,858,781); Soybean variety 91M10 (U.S. Pat. No. 6,958,437);Soybean variety 92M10 (U.S. Pat. No. 6,916,975); Soybean variety XB10G03(U.S. Pat. No. 6,806,405); Soybean variety 92M31 (U.S. Pat. No.6,846,974); Soybean variety XB38D03 (U.S. Pat. No. 6,806,404); Soybeanvariety XB34N03 (U.S. Pat. No. 6,803,508); Soybean variety XB30W03 (U.S.Pat. No. 6,809,236); Soybean variety XB37J03 (U.S. Pat. No. 6,844,488);Soybean variety SE72581 (US2004148665); Soybean variety SE90076(US2004148664); Soybean variety SD82997 (US2004148663); Soybean variety0037393 (US2004148662); Soybean variety 0088414 (US2004148661); Soybeanvariety 0149926 (US2004148660); Soybean variety 0037209 (US2004148659);Soybean variety 93B36 (U.S. Pat. No. 6,833,498); Soybean variety 90B74(U.S. Pat. No. 6,812,384); Soybean variety 90B51 (U.S. Pat. No.6,818,809); Soybean variety 91B03 (U.S. Pat. No. 6,815,584); Soybeanvariety 95B43 (U.S. Pat. No. 6,818,808); Soybean variety 95B42 (U.S.Pat. No. 6,815,583); Soybean variety 92B47 (U.S. Pat. No. 6,812,383);Soybean variety SE90346 (US2004055055); Soybean variety 0007583(US2004010824); Soybean variety 0008079 (US2004010823); Soybean variety502-AP98041-2-333-01 (US2003121076); Soybean variety S02-98041-2-251-01(US2003182694); Soybean variety S02-AP98041-2-262-02 (US2003196220);Soybean variety S02-95021-55-240-BA (US2003188348); Soybean varietyAPA94-31572 (US2003061641); Soybean variety AP98041-1-203(US2003056251); Soybean variety APA95-15294 (US2003061640); Soybeanvariety AP98041-4-117 (US2003056250); Soybean variety 91B33 (U.S. Pat.No. 6,580,018); Soybean variety 93B85 (U.S. Pat. No. 6,605,762); Soybeanvariety 92B76 (U.S. Pat. No. 6,610,911); Soybean variety 92B38 (U.S.Pat. No. 6,605,761); Soybean variety 94B24 (U.S. Pat. No. 6,613,967);Soybean variety 94B73 (U.S. Pat. No. 6,605,760); Soybean variety 93B86(U.S. Pat. No. 6,610,910); Soybean variety 91B12 (U.S. Pat. No.6,583,343); Soybean variety 95B34 (U.S. Pat. No. 6,605,759); Soybeanvariety 94B23 (U.S. Pat. No. 6,605,758); Soybean variety 90B11 (U.S.Pat. No. 6,583,342); Soybean variety 91B92 (U.S. Pat. No. 6,586,659);Soybean variety 95B96 (U.S. Pat. No. 6,605,757); Soybean variety 93B72(U.S. Pat. No. 6,566,589); Soybean variety 95B97 (U.S. Pat. No.6,613,966); Soybean variety 92B95 (U.S. Pat. No. 6,608,243); Soybeanvariety 93B47 (U.S. Pat. No. 6,583,341); Soybean variety 97B52 (U.S.Pat. No. 6,605,756); Soybean variety 93B15 (U.S. Pat. No. 6,617,499);Soybean variety 94B54 (U.S. Pat. No. 6,613,965); Soybean variety 93B67(U.S. Pat. No. 6,573,433); Soybean variety 93B87 (U.S. Pat. No.6,727,410); Soybean variety 96B51 (U.S. Pat. No. 6,613,964); Soybeanvariety 92B84 (U.S. Pat. No. 6,730,829); Soybean variety 92B12 (U.S.Pat. No. 6,605,755); Soybean variety 90A07 (U.S. Pat. No. 6,320,105);Soybean variety 93B26 (U.S. Pat. No. 6,342,659); Soybean variety 96B21(U.S. Pat. No. 6,369,301); Soybean variety 92B63 (U.S. Pat. No.6,326,529); Soybean variety 93B46 (U.S. Pat. No. 6,323,402); Soybeanvariety 92B75 (U.S. Pat. No. 6,362,400); Soybean variety 93B08 (U.S.Pat. No. 6,323,401); Soybean variety 97B62 (U.S. Pat. No. 6,323,400);Soybean variety 92B37 (U.S. Pat. No. 6,323,399); Soybean variety 92B56(U.S. Pat. No. 6,339,186); Soybean variety 93B66 (U.S. Pat. No.6,307,131); Soybean variety 92B62 (U.S. Pat. No. 6,346,657); Soybeanvariety 92B36 (U.S. Pat. No. 6,369,300); Soybean variety 90B73 (U.S.Pat. No. 6,316,700); Soybean variety 95B95 (U.S. Pat. No. 6,323,398);Soybean variety 93B65 (U.S. Pat. No. 6,229,074); Soybean variety 92B24(U.S. Pat. No. 6,284,950); Soybean variety 94B53 (U.S. Pat. No.6,235,976); Soybean variety 94B22 (U.S. Pat. No. 6,140,557); Soybeanvariety 93B84 (U.S. Pat. No. 6,143,956); Soybean variety 95B32 (U.S.Pat. No. 6,229,073); Soybean variety 95B53 (U.S. Pat. No. 6,147,283);Soybean variety 93B35 (U.S. Pat. No. 6,153,816); Soybean variety 93B07(U.S. Pat. No. 6,143,955); Soybean variety 92B74 (U.S. Pat. No.6,124,526); Soybean variety 92B35 (U.S. Pat. No. 6,166,296); Soybeanvariety 94B45 (U.S. Pat. No. 6,162,968); Soybean variety 96B01 (U.S.Pat. No. 6,143,954); Soybean variety 93B53 (U.S. Pat. No. 6,335,197).

It is observed that the introgression of the elite events into thesecultivars does not significantly influence any of the desirablephenotypic or agronomic characteristics of these cultivars (no yielddrag) while expression of the transgene, as determined by glyphosateand/or isoxaflutole or glufosinate tolerance, meets commerciallyacceptable levels.

The stacks may be advantageously further combined with one or more othersoybean events available in the market, including but not limited toother herbicide tolerance events, such as events described in USDA-APHISpetitions: 09-349-01p, 09-201-01p, 09-183-01p, 09-082-01p, 09-015-01p,06-354-01p, 06-271-01p, 06-178-01p, 98-238-01p, 98-014-01p, 97-008-01p,96-068-01p, 95-335-01p, 93-258-01p, (see, e.g., the USDA's aphiswebsite) or event MON89788 (Glyphosate tolerance) described in US2006-282915, event DP-305423-1 (High oleic acid/ALS inhibitor tolerance)described in WO 2008/054747, MON87701 described in US2009130071, event3560.4.3.5 described in US2009036308, or event DP-305423-1 described inUS2008312082, or event BPS-CV127-9 (Event 127) of WO 2010/080829.

As used in the claims below, unless otherwise clearly indicated, theterm “plant” is intended to encompass plant tissues, at any stage ofmaturity, as well as any cells, tissues, or organs taken from or derivedfrom any such plant, including without limitation, any seeds, leaves,stems, flowers, roots, single cells, gametes, cell cultures, tissuecultures or protoplasts.

Reference seed comprising elite event EE-GM3 was deposited as32-RRMM-0531 at the NCIMB (Ferguson Building, Craibstone Estate,Bucksburn, Aberdeen AB9YA, Scotland) on Oct. 12, 2009, under NCIMBaccession number NCIMB 41659, and the viability thereof was confirmed.All restrictions on the availability to the public of the depositedmaterial will be irrevocably removed upon the granting of a patent onthe present application. An alternative name for EE-GM3 is event FG-072,or MST-FGØ72-3.

Reference seed comprising elite event EE-GM1 was deposited as32RR1VM0368 at the NCIMB (Ferguson Building, Craibstone Estate,Bucksburn, Aberdeen AB9YA, Scotland) on Oct. 12, 2009, under NCIMBaccession number NCIMB 41658. All restrictions on the availability tothe public of the deposited material will be irrevocably removed uponthe granting of a patent on the present application. An alternative namefor EE-GM1 is LL27, A2704-12, or ACS-GM ØØ5-3.

Reference seed comprising elite event EE-GM2 was deposited as 32CON0688at the NCIMB (Ferguson Building, Craibstone Estate, Bucksburn, AberdeenAB9YA, Scotland) on Oct. 12, 2009, under NCIMB accession number NCIMB41660. All restrictions on the availability to the public of thedeposited material will be irrevocably removed upon the granting of apatent on the present application. An alternative name for EE-GM2 isLL55, A5547-127, or ACS-GM ØØ6-4.

Reference seed comprising elite event EE-GM3 and EE-GM1 was deposited as111606 soybean at the ATCC (American Type Culture Collection, 10801University Blvd., Manassas, Va. 20110-2209, USA) on Jun. 11, 2010, underATCC accession number PTA-11041. All restrictions on the availability tothe public of the deposited material will be irrevocably removed uponthe granting of a patent on the present application.

Reference seed comprising elite event EE-GM3 and EE-GM2 was deposited as05SHX2XEB Soybean at the ATCC (American Type Culture Collection, 10801University Blvd., Manassas, Va. 20110-2209, USA) on Jun. 11, 2010, underATCC accession number PTA-11042. All restrictions on the availability tothe public of the deposited material will be irrevocably removed uponthe granting of a patent on the present application.

The above description of the invention is intended to be illustrativeand not limiting.

Various changes or modifications in the embodiments described may occurto those skilled in the art. These can be made without departing fromthe spirit or scope of the invention.

The invention claimed is:
 1. A plant, cell, plant part, tissue, seed, orprogeny thereof, which is (a) a soybean plant, cell, a plant part,tissue, seed, or progeny thereof, each comprising elite event EE-GM3 andelite event EE-GM1, reference seed comprising said elite event EE-GM3having been deposited at the NCIMB under deposit number NCIMB 41659 andreference seed comprising said elite event EE-GM1 having being depositedat the NCIMB under deposit number NCIMB 41658, or reference seedcomprising elite event EE-GM3 and elite event EE-GM1 in its genomehaving been deposited at the ATCC under deposit number PTA-11041; or (b)a soybean plant, plant part, seed, cell, or tissue thereof, eachcomprising elite event EE-GM3 and elite event EE-GM1, said soybean plantbeing obtained by crossing a soybean plant comprising elite event EE-GM3obtainable by propagation of, breeding with, or propagation of andbreeding with a soybean plant grown from the seed deposited at the NCIMBunder deposit number NCIMB 41659 with a soybean plant comprising eliteevent EE-GM1 in its genome, obtainable by propagation of, breeding with,or propagation of and breeding with a soybean plant grown from the seeddeposited at the NCIMB under deposit number NCIMB 41658, or said soybeanplant being obtainable by propagation of, breeding with, or propagationof and breeding with a soybean plant grown from the seed deposited atthe ATCC under accession number PTA-11041; or (c) a soybean seedcomprising elite events EE-GM3 and EE-GM1, reference seed comprisingelite event EE-GM3 having been deposited at the NCIMB under depositnumber NCIMB 41659, reference seed comprising elite event EE-GM1 havingbeen deposited at the NCIMB under deposit number NCIMB 41658, andreference seed comprising elite events EE-GM3 and EE-GM1 having beendeposited at the ATCC under deposit number PTA-11041; or (d) a soybeanplant, plant part, cell, or tissue, each comprising elite events EE-GM3and EE-GM1, obtainable from the seed of (c); or (e) a soybean plant, orcells, part, seed or progeny thereof, each comprising elite eventsEE-GM3 and EE-GM1 , wherein the inserted foreign DNA of elite eventEE-GM3 is oriented as indicated in FIG. 1, and wherein said event has 5′and 3′ flanking DNA and sequences in the foreign DNA contiguoustherewith, wherein said 5′ flanking DNA and the foreign DNA contiguoustherewith comprises a nucleotide sequence of SEQ ID No: 2, and said 3′flanking DNA and the foreign DNA contiguous therewith comprises anucleotide sequence of SEQ ID No: 3, said inserted foreign DNA of FIG. 1being present in the soybean elite event EE-GM3 deposited under depositnumber NCIMB 41659, and wherein the inserted foreign DNA of elite eventEE-GM1 is oriented as indicated in FIG. 3, and wherein said event has 5′and 3′ flanking DNA and sequences in the foreign DNA contiguoustherewith, wherein said 5′ flanking DNA and the foreign DNA contiguoustherewith comprises a nucleotide sequence of SEQ ID No: 12, and said 3′flanking DNA and the foreign DNA contiguous therewith comprises anucleotide sequence of SEQ ID No: 13, said inserted foreign DNA of FIG.3 being present in the soybean elite event EE-GM2 deposited underdeposit number NCIMB 41658, or (f) a soybean plant, plant cell, tissue,or seed, each comprising in their genome a nucleic acid moleculecomprising a nucleotide sequence of SEQ ID No: 1 from nucleotideposition 188 to nucleotide position 7252 or the complement thereof, andcomprising in their genome a nucleic acid molecule comprising anucleotide sequence of elite event EE-GM1 or the complement thereof,reference seed comprising elite event EE-GM1 having been deposited underdeposit number NCIMB 41658, wherein said nucleotide sequence of eliteevent EE-GM1 comprises or is the foreign DNA sequence in SEQ ID No: 12or 13, or the foreign DNA sequence in the plant genome between thesequence of SEQ ID No: 12 and that of SEQ ID No:
 13. 2. The soybeanplant, seed, cell, tissue, part or progeny of claim 1(a), the genomicDNA of which, when amplified with two primers comprising the nucleotidesequence of SEQ ID No: 4 and SEQ ID No: 5 respectively, yields a DNAfragment of about 263 bp and the genomic DNA of which, when amplifiedwith two primers comprising the nucleotide sequence of SEQ ID No: 16 andSEQ ID No: 17 respectively, yields a DNA fragment of about 183 bp.
 3. Amethod for producing a soybean plant or seed comprising elite eventsEE-GM3 and EE-GM1 comprising crossing a plant according to claim 1(a)with another soybean plant, and planting the seed comprising said eliteevents obtained from said cross.
 4. A soybean plant, cell, plant part,seed, or progeny thereof, each comprising in its genome elite eventEE-GM3, wherein said event comprises in order the following nucleotidesequences: (a) the nucleotide sequence of SEQ ID No: 2 from nucleotide 1to 1451; (b) the nucleotide sequence of the complement of the nucleotidesequence of SEQ ID No: 1 from nucleotide 6760 to nucleotide 6958; (c)the nucleotide sequence of SEQ ID No: 1 from nucleotide 6874 tonucleotide 7298; (d) the nucleotide sequence of SEQ ID No: 1 fromnucleotide 7 to nucleotide 7291; (e) the nucleotide sequence of SEQ IDNo: 1 from nucleotide 12 to nucleotide 7265; (f) the nucleotide sequenceof SEQ ID No: 3 from nucleotide 217 to nucleotide 240; (g) thenucleotide sequence of SEQ ID No: 3 from nucleotide 241 to nucleotide1408; and further comprising in its genome elite event EE-GM1, whereinsaid event comprises in order the following sequences: (h) thenucleotide sequence of SEQ ID No: 12 from nucleotide 1 to 209; (i) thenucleotide sequence of SEQ ID No: 11 from nucleotide 340 to nucleotide3461; (j) the nucleotide sequence of the complement of the nucleotidesequence of SEQ ID No: 11 from nucleotide 1 to nucleotide 336; (k) thenucleotide sequence of the complement of the nucleotide sequence of SEQID No: 11 from nucleotide 3462 to nucleotide 4076; (l) the nucleotidesequence of SEQ ID No: 11 from nucleotide 337 to nucleotide 3043; (m)the nucleotide sequence of SEQ ID No: 13 from nucleotide 559 tonucleotide 568; and (n) the nucleotide sequence of SEQ ID No: 13 fromnucleotide 569 to nucleotide
 1000. 5. A plant, cell, plant part, tissue,seed, or progeny thereof, which is (a) a soybean plant, cell, plantpart, tissue, seed, or progeny thereof, each comprising elite eventEE-GM3 and elite event EE-GM2 in its genome, reference seed comprisingsaid elite event EE-GM3 having been deposited at the NCIMB under depositnumber NCIMB 41659 and reference seed comprising said elite event EE-GM2having been deposited at the NCIMB under deposit number NCIMB 41660, orreference seed comprising elite event EE-GM3 and elite event EE-GM2 inits genome having been deposited at the ATCC under deposit numberPTA-11042; or (b) a soybean plant, plant part, tissue, seed or progenythereof, each comprising elite event EE-GM3 and elite event EE-GM2 inits genome, said soybean plant being obtained by crossing a soybeanplant comprising elite event EE-GM3 in its genome, obtainable bypropagation of, breeding with, or propagation of and breeding with asoybean plant grown from the seed deposited at the NCIMB under depositnumber NCIMB 41659 with a soybean plant comprising elite event EE-GM2 inits genome, obtainable by propagation of, breeding with, or propagationof and breeding with a soybean plant grown from the seed deposited atthe NCIMB under deposit number NCIMB 41660, or said soybean plant beingobtainable by propagation of, breeding with, or propagation of andbreeding with a soybean plant grown from the seed deposited at the ATCCunder accession number PTA-11042; or (c) a soybean seed comprising eliteevents EE-GM3 and EE-GM2, reference seed comprising elite event EE-GM3having been deposited at the NCIMB under deposit number NCIMB 41659,reference seed comprising elite event EE-GM2 having been deposited atthe NCIMB under deposit number NCIMB 41660, and reference seedcomprising elite events EE-GM3 and EE-GM2 having been deposited at theATCC under deposit number PTA-11042; or (d) a soybean plant, cell, plantpart, tissue, or progeny therof, each comprising elite events EE-GM3 andEE-GM2, produced from the seed of (c); or (e) a soybean plant, cell,plant part, tissue, seed, or progeny thereof, each comprising eliteevents EE-GM3 and EE-GM1, wherein said events comprise a nucleic acidsequence of SEQ ID No: 2 from nucleotide 1431 to 1472 and a nucleic acidsequence of SEQ ID No: 3 from nucleotide 220 to 261, or the complementof said sequences, and also a nucleic acid sequence of SEQ ID No: 12from nucleotide 199 to 220 and a nucleic acid sequence of SEQ ID No: 13from nucleotide 558 to 579, or the complement of said sequences; or (f)a soybean plant, cell, plant part, tissue, seed, or progeny thereof,each comprising elite events EE-GM3 and EE-GM2, wherein said eventscomprise a nucleic acid sequence of SEQ ID No: 2 from nucleotide 1431 to1472 and a nucleic acid sequence of SEQ ID No: 3 from nucleotide 220 to261, or the complement of said sequences, and also a nucleic acidsequence of SEQ ID No: 14 from nucleotide 301 to 322 and a nucleic acidsequence of SEQ ID No: 15 from nucleotide 497 to 518, or the complementof said sequences; or (g) a soybean plant, cell, plant part, tissue,seed, or progeny therof, each comprising in their genome a nucleic acidmolecule comprising a nucleotide sequence of SEQ ID No: 20 fromnucleotide position 1452 to nucleotide position 16638 or the complementthereof, or a nucleotide sequence of SEQ ID No: 20 or the complementthereof, and comprising in their genome a nucleic acid moleculecomprising a nucleotide sequence of elite events EE-GM1 or EE-GM2 or thecomplement thereof, reference seed comprising elite event EE-GM1 havingbeen deposited under deposit number NCIMB 41658, and reference seedcomprising elite event EE-GM2 having been deposited under deposit numberNCIMB 41660, wherein said nucleotide sequence of elite events EE-GM1 orEE-GM2 comprises or is the foreign DNA sequence in SEQ ID No: 12 or 13,or the foreign DNA sequence in SEQ ID No: 14 or 15, respectively, or theforeign DNA sequence in the plant genome between the sequence of SEQ IDNo: 12 and that of SEQ ID No: 13, or the foreign DNA sequence in theplant genome between the sequence of SEQ ID No: 14 and that of SEQ IDNo: 15, respectively; or (h) a soybean plant, cell, plant part, tissue,seed, or progeny thereof, each comprising elite events EE-GM3 and EE-GM2in its genome, wherein the inserted foreign DNA of elite event EE-GM3 isoriented as indicated in FIG. 1, and wherein said event has 5′ and 3′flanking DNA and foreign DNA contiguous therewith, wherein said 5′flanking DNA and the foreign DNA contiguous therewith comprises anucleotide sequence of SEQ ID No: 2, and said 3′ flanking DNA and theforeign DNA contiguous therewith comprises a nucleotide sequence of SEQID No: 3, said inserted foreign DNA of FIG. 1 being present in thesoybean elite event EE-GM3 deposited under deposit number NCIMB 41659,and wherein the inserted foreign DNA of elite event EE-GM2 is orientedas indicated in FIG. 4, and wherein said event has 5′ and 3′ flankingDNA and foreign DNA contiguous therewith, wherein said 5′ flanking DNAand the foreign DNA contiguous therewith comprises a nucleotide sequenceof SEQ ID No: 14 and said 3′ flanking DNA and the foreign DNA contiguoustherewith comprises a nucleotide sequence of SEQ ID No: 15, saidinserted foreign DNA of FIG. 4 being present in the soybean elite eventEE-GM2 deposited under deposit number NCIMB 41660, or (i) a soybeanplant, cell, plant part, tissue, seed, or progeny thereof, eachcomprising in their genome a nucleic acid molecule comprising anucleotide sequence of SEQ ID No: 1 from nucleotide position 188 tonucleotide position 7252 or the complement thereof, and comprising intheir genome a nucleic acid molecule comprising a nucleotide sequence ofelite event EE-GM2 or the complement thereof, reference seed comprisingelite event EE-GM2 having been deposited under deposit number NCIMB41660, wherein said nucleotide sequence of elite event EE-GM2 comprisesor is the foreign DNA sequence in SEQ ID No: 14 or 15, or the foreignDNA sequence in the plant genome between the sequence of SEQ ID No: 14and that of SEQ ID No:
 15. 6. The soybean plant, cell, plant part,tissue, seed, or progeny of claim 5(a), the genomic DNA of which, whenamplified with two primers comprising the nucleotide sequence of SEQ IDNo: 4 and SEQ ID No: 5 respectively, yields a DNA fragment of about 263by and the genomic DNA of which, when amplified with two primerscomprising the nucleotide sequence of SEQ ID No: 18 and SEQ ID No: 19respectively, yields a DNA fragment of about 151 bp.
 7. A method forproducing a soybean plant or seed comprising elite events EE-GM3 andEE-GM2 said method comprising crossing a plant according to claim 5(a)with another soybean plant, and planting the seed comprising said eliteevents obtained from said cross.
 8. A soybean plant, cell, plant part,seed, or progeny thereof, each comprising elite event EE-GM3, whereinsaid event comprises in order the following nucleotide sequences: (a)the nucleotide sequence of SEQ ID No: 2 from nucleotide 1 to 1451; (b)the nucleotide sequence of the complement of the nucleotide sequence ofSEQ ID No: 1 from nucleotide 6760 to nucleotide 6958; (c) the nucleotidesequence of SEQ ID No: 1 from nucleotide 6874 to nucleotide 7298; (d)the nucleotide sequence of SEQ ID No: 1 from nucleotide 7 to nucleotide7291; (e) the nucleotide sequence of SEQ ID No: 1 from nucleotide 12 tonucleotide 7265; (f) the nucleotide sequence of SEQ ID No: 3 fromnucleotide 217 to nucleotide 240; and (g) the nucleotide sequence of SEQID No: 3 from nucleotide 241 to nucleotide 1408, and further comprisingin its genome elite event EE-GM2, wherein said event comprises in orderthe following sequences: (h) the nucleotide sequence of SEQ ID No: 14from nucleotide 1 to 311; (i) the nucleotide sequence of SEQ ID No: 11from nucleotide 3458 to nucleotide 3848; (j) the nucleotide sequence ofSEQ ID No: 11 from nucleotide 413 to nucleotide 3457; (k) the nucleotidesequence of SEQ ID No: 15 from nucleotide 508 to nucleotide
 1880. 9. Thesoybean plant, cell, part, tissue, seed or progeny of claim 1(a). 10.The soybean plant, cell, plant part, tissue, seed, or progeny of claim5(a).
 11. The soybean plant, cell, plant part, tissue, seed, or progenyof claim 5(a), wherein said elite event EE-GM3 comprises a foreign DNAcomprising a chimeric gene encoding the 4-hydroxyphenylpyruvatedioxygenase of Pseudomonas fluorescens strain A32 modified by thereplacement of the amino acid Glycine 336 with a Tryptophane (HPPD PFW366 gene) and a chimeric gene encoding the 5-enolpyruvylshikimate-3-phosphate synthase enzyme double mutant from Zea Mays(2mEPSPS gene), said elite event EE-GM3 also comprising nucleotides 1 to1451 of SEQ ID No: 2 immediately upstream of and contiguous with saidforeign DNA and nucleotides 241 to 1408 of SEQ ID No: 3 immediatelydownstream of and contiguous with said foreign DNA, and wherein saidelite event EE-GM2 comprises a foreign DNA comprising a chimericphosphinothricin acetyltransferase encoding gene, said elite eventEE-GM2 also comprising nucleotides 1 to 311 of SEQ ID No: 14 immediatelyupstream of and contiguous with said foreign DNA and nucleotides 508 to1880 of SEQ ID No: 15 immediately downstream of and contiguous with saidforeign DNA.
 12. The soybean plant, cell, plant part, tissue, seed, orprogeny of claim 5(h).
 13. The soybean seed of claim 5(c).
 14. Thesoybean plant, cell, plant part, tissue, seed, or progeny of claim 5(d).15. The soybean plant, cell, plant part, tissue, seed, or progeny ofclaim 5(e).
 16. The soybean plant, cell, plant part, tissue, seed, orprogeny of claim 5(f).
 17. The soybean plant, cell, plant part, tissue,seed, or progeny of claim 5(g).
 18. The soybean plant, cell, plant part,tissue, seed, or progeny of claim 5(i), comprising in their genome anucleic acid molecule comprising a nucleotide sequence of SEQ ID No: 20or SEQ ID No: 20 from nucleotide position 1452 to nucleotide position16638 or the complement thereof.
 19. A method for producing a soybeanplant or seed comprising elite events EE-GM3 and EE-GM2 comprisingcrossing a plant according to claim 5(h) with another soybean plant, andplanting the seed comprising elite events EE-GM3 and EE-GM2 obtainedfrom said cross.
 20. A method for producing a soybean plant or seedcomprising elite events EE-GM3 and EE-GM2 comprising crossing a plantaccording to claim 5(f) with another soybean plant, and planting theseed comprising elite events EE-GM3 and EE-GM2 obtained from said cross.21. A soybean product produced from seed comprising elite events EE-GM3and EE-GM2 of any one of claim 5(a), 5(h) or 5(f), wherein said soybeanproduct is or comprises soybean meal, ground seeds, flour, or flakes.22. A soybean product produced from seed comprising elite events EE-GM3and EE-GM2 of claim 6, wherein said soybean product is or comprisessoybean meal, ground seeds, flour, or flakes.
 23. A soybean productproduced from seed comprising elite events EE-GM3 and EE-GM2 of claim 8,wherein said soybean product is or comprises soybean meal, ground seeds,flour, or flakes.
 24. A soybean product produced from seed comprisingelite events EE-GM3 and EE-GM2 of claim 18, wherein said soybean productis or comprises soybean meal, ground seeds, flour, or flakes.
 25. Amethod for producing a soybean product, comprising obtaining soybeanseed comprising elite event EE-GM3 and elite event EE-GM2 of claim 5(a),and producing such soybean product therefrom.
 26. A method for producinga soybean product, comprising obtaining soybean seed comprising eliteevent EE-GM3 and elite event EE-GM2 of claim 5(h), and producing suchsoybean product therefrom.
 27. A method for producing a soybean product,comprising obtaining soybean seed comprising elite event EE-GM3 andelite event EE-GM2 of claim 5(f), and producing such soybean producttherefrom.
 28. A method for controlling weeds in a field of soybeanplants comprising elite events EE-GM3 and EE-GM2 of claim 5(a),comprising treating the field with an effective amount of anisoxaflutole-based and/or glyphosate-based and/or glufosinate-basedherbicide, wherein such plants are tolerant to such herbicide(s).
 29. Aprocess for weed control, comprising treating a field in which seedscontaining elite events EE-GM3 and EE-GM2 of claim 5(a) were sown withan HPPD inhibitor herbicide, optionally followed by application ofglyphosate and/or glufosinate or a mixture of an HPPD inhibitor withglyphosate and/or glufosinate as post-emergent herbicide over the top ofthe plants.
 30. A process for weed control, comprising treating soybeanplants containing elite events EE-GM3 and EE-GM2 of claim 5(a) with anHPPD inhibitor herbicide over the top of the plants, applied together,followed by or preceded by a treatment with glyphosate and/orglufosinate as post-emergent herbicide over the top of the plants.
 31. Amethod for producing a soybean plant tolerant to glyphosate and/orglufosinate and/or isoxaflutole herbicides, comprising introducing intothe genome of such plant elite event EE-GM3 and elite event EE-GM2 ofclaim 5(a).
 32. A method for controlling weeds in a field of soybeanplants comprising elite events EE-GM3 and EE-GM1 of claim 1(a)comprising treating the field with an effective amount of anisoxaflutole-based and/or glyphosate-based and/or glufosinate-basedherbicide, wherein such plants are tolerant to such herbicide(s).
 33. Aprocess for weed control, comprising treating a field in which seedscontaining elite events EE-GM3 and EE-GM1 of claim 1(a) were sown withan HPPD inhibitor herbicide, optionally followed by application ofglyphosate and/or glufosinate or a mixture of an HPPD inhibitor withglyphosate and/or glufosinate as post-emergent herbicide over the top ofthe plants.
 34. A process for weed control, comprising treating soybeanplants containing elite events EE-GM3 and EE-GM1 of claim 1(a) with anHPPD inhibitor herbicide over the top of the plants, applied together,followed by or preceded by a treatment with glyphosate and/orglufosinate as post-emergent herbicide over the top of the plants.
 35. Amethod for producing a soybean plant tolerant to glyphosate and/orglufosinate and/or isoxaflutole herbicides, comprising introducing intothe genome of such plant elite event EE-GM3 and elite event EE-GM1 ofclaim 1(a).
 36. A soybean plant, plant part, cell, tissue, or seedcomprising elite events EE-GM3 and EE-GM1.
 37. A method for producing asoybean plant or seed comprising elite events EE-GM3 and EE-GM1, saidmethod comprising crossing the plant according to claim 36 with anothersoybean plant, and planting the seed comprising elite events EE-GM3 andEE-GM1 obtained from said cross.
 38. A soybean product produced from theseed of claim 36, wherein said soybean product comprises elite eventsEE-GM3 and EE-GM1 and comprises soybean meal, ground seeds, soybeanflour, or soybean flakes.
 39. A method for producing a soybean planttolerant to glyphosate and/or isoxaflutole herbicides, comprisingintroducing into the genome of said soybean plant elite events EE-GM3and EE-GM1 of claim 36, and selecting a progeny plant tolerant toglyphosate and/or isoxaflutole, wherein said progeny plant compriseselite events EE-GM3 and EE-GM1.
 40. A method for controlling weeds in afield of the soybean plant of claim 36, comprising treating the fieldwith an effective amount of an isoxaflutole-based, glyphosate-basedand/or glufosinate-based herbicide, wherein said soybean plant istolerant to said isoxaflutole-based herbicide.
 41. A method forcontrolling weeds in a field of the soybean plants or seeds of claim 36,comprising treating a field with an HPPD inhibitor herbicide, beforesaid soybean plants are planted or the seeds are sown, followed byplanting or sowing of said soybeans in that same pre-treated field. 42.The method of claim 41, wherein glyphosate, or an HPPDinhibitor-glyphosate mixture, is applied as post-emergent herbicide overthe top of said soybean plants once weeds tend to re-appear.
 43. Amethod for controlling weeds in a field of the soybean seeds of claim36, comprising treating a field in which said seeds were sown, with anHPPD inhibitor herbicide before the soybean plants emerge but after theseeds are sown.
 44. The method of claim 43, wherein glyphosate, or anHPPD inhibitor-glyphosate mixture, is applied as post-emergent herbicideover the top of said soybean plants once weeds tend to re-appear.
 45. Amethod for controlling weeds in a field of the soybean plants of claim36, comprising treating said plants with an HPPD inhibitor herbicideover the top of the soybean plants, which application can be togetherwith, followed by or preceded by a treatment with glyphosate aspost-emergent herbicide over the top of the plants.
 46. A soybean plant,plant part, cell, tissue, or seed comprising elite events EE-GM3 andEE-GM2.
 47. A method for producing a soybean plant or seed comprisingelite events EE-GM3 and EE-GM2, said method comprising crossing theplant according to claim 46 with another soybean plant, and planting theseed comprising elite events EE-GM3 and EE-GM2 obtained from said cross.48. A soybean product produced from the seed of claim 46, wherein saidsoybean product comprises elite events EE-GM3 and EE-GM2 and comprisessoybean meal, ground seeds, soybean flour, or soybean flakes.
 49. Amethod for producing a soybean plant tolerant to glyphosate and/orisoxaflutole herbicides, comprising introducing into the genome of saidsoybean plant elite events EE-GM3 and EE-GM2 of claim 46, and selectinga progeny plant tolerant to glyphosate and/or isoxaflutole, wherein saidprogeny plant comprises elite events EE-GM3 and EE-GM2.
 50. A method forcontrolling weeds in a field of the soybean plant of claim 46,comprising treating the field with an effective amount of anisoxaflutole-based herbicide, wherein said soybean plant is tolerant tosaid isoxaflutole-based herbicide.
 51. A method for controlling weeds ina field of the soybean plants or seeds of claim 46, comprising treatinga field with an HPPD inhibitor herbicide, before said soybean plants areplanted or the seeds are sown, followed by planting or sowing of saidsoybeans in that same pre-treated field.
 52. The method of claim 51,wherein glyphosate, or an HPPD inhibitor-glyphosate mixture, is appliedas post-emergent herbicide over the top of said soybean plants onceweeds tend to re-appear.
 53. A method for controlling weeds in a fieldof the soybean seeds of claim 46, comprising treating a field in whichsaid seeds were sown, with an HPPD inhibitor herbicide before thesoybean plants emerge but after the seeds are sown.
 54. The method ofclaim 53, wherein glyphosate, or an HPPD inhibitor-glyphosate mixture,is applied as post-emergent herbicide over the top of said soybeanplants once weeds tend to re-appear.
 55. A method for controlling weedsin a field of the soybean plants of claim 46, comprising treating saidplants with an HPPD inhibitor herbicide over the top of the soybeanplants, which application can be together with, followed by or precededby a treatment with glyphosate as post-emergent herbicide over the topof the plants.
 56. The soybean plant of claim 1(a), treated with any oneof the following insecticides, herbicides or fungicides: (a) SoybeanHerbicides: Alachlor, Bentazone, Trifluralin, Chlorimuron-Ethyl,Cloransulam-Methyl, Fenoxaprop, Fomesafen, Fluazifop, Glyphosate,Imazamox, Imazaquin, Imazethapyr, (S-)Metolachlor, Metribuzin,Pendimethalin, Tepraloxydim, Isoxaflutole, or Glufosinate; (b) SoybeanInsecticides: Lambda-cyhalothrin, Methomyl, Parathion, Thiocarb,Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid,Dinetofuran, Flubendiamide, Rynaxypyr, Cyazypyr, Spinosad, Spinotoram,Emamectin-Benzoate, Fipronil, Ethiprole, Deltamethrin, β-Cyfluthrin,gamma and lambda Cyhalothrin, 4-[[(6Chlorpyridin-3-yl)methyl](2,2-difluorethy)amino]furan-2(5H)-on,Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb, orbeta-Cyfluthrin; (c) Soybean Fungicides: Azoxystrobin, Cyproconazole,Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole,Trifloxystrobin, Prothioconazole, or Tetraconazole.
 57. The soybeanplant of claim 5(a), treated with any one of the following insecticides,herbicides or fungicides: (a) Soybean Herbicides: Alachlor, Bentazone,Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl, Fenoxaprop,Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin, Imazethapyr,(S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim, Isoxaflutole,or Glufosinate; (b) Soybean Insecticides: Lambda-cyhalothrin, Methomyl,Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole,Deltamethrin, β-Cyfluthrin, gamma and lambda Cyhalothrin,4-[[(6-Chlorpyridin -3-yl)methyl](2,2-difluorethy)amino]furan-2(5H)-on,Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb, orbeta-Cyfluthrin; (c) Soybean Fungicides: Azoxystrobin, Cyproconazole,Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole,Trifloxystrobin, Prothioconazole, or Tetraconazole.
 58. The soybeanplant of claim 36, treated with any one of the following insecticides,herbicides or fungicides: (a) Soybean Herbicides: Alachlor, Bentazone,Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl, Fenoxaprop,Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin, Imazethapyr,(S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim, Isoxaflutole,or Glufosinate; (b) Soybean Insecticides: Lambda-cyhalothrin, Methomyl,Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole,Deltamethrin,β-Cyfluthrin, gamma and lambda Cyhalothrin,4-[[(6-Chlorpyridin -3-yl)methyl](2,2-difluorethy)amino]furan-2(5H)-on,Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb, orbeta-Cyfluthrin; (c) Soybean Fungicides: Azoxystrobin, Cyproconazole,Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole,Trifloxystrobin, Prothioconazole, or Tetraconazole.
 59. The soybeanplant of claim 46, treated with any one of the following insecticides,herbicides or fungicides: (a) Soybean Herbicides: Alachlor, Bentazone,Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl, Fenoxaprop,Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin, Imazethapyr,(S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim, Isoxaflutole,or Glufosinate; (b) Soybean Insecticides: Lambda-cyhalothrin, Methomyl,Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole,Deltamethrin, β-Cyfluthrin, gamma and lambda Cyhalothrin,4-[[(6-Chlorpyridin -3-yl)methyl](2,2-difluorethyeamino]furan-2(5H)-on,Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb, orbeta-Cyfluthrin; (c) Soybean Fungicides: Azoxystrobin, Cyproconazole,Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole,Trifloxystrobin, Prothioconazole, or Tetraconazole.
 60. The soybeanplant, or cell, part, tissue, seed or progeny of claim 1(a).
 61. Thesoybean plant, plant part, or seed, cell or tissue of claim 1(b). 62.The soybean seed of claim 1(c).
 63. The soybean plant, plant part, cell,or tissue of claim 1(d).
 64. The soybean plant, or cells, part, seed orprogeny of claim 1(e).
 65. The soybean plant, plant cell, tissue, orseed of claim 1(f).